Compositions and Methods for Treating Non-Alcoholic Steatohepatitis

ABSTRACT

The disclosure provides for a method for treating a fatty liver disorder in a subject in need thereof, comprising selecting a subject having or suspected of having a fatty liver disease or disorder, wherein the subject is non diabetic, pre-diabetic, mildly diabetic, or has normal or substantially normal biliary tract function; and administering a therapeutically effective amount of a pharmaceutical composition comprising ethyl eicosapentanoate (EPA-E). In some cases EPA-E present may be at least 40% by weight in total of the fatty acids and their derivatives.

CROSS-REFERENCE

This application claims the benefit of U.S. Provisional Application No.61/791,533, filed Mar. 15, 2013; and 61/793,309, filed Mar. 15, 2013;each of which applications is incorporated herein in its entirety byreference.

BACKGROUND

Heavy alcohol use is known to lead to liver complications, includingalcoholic hepatitis which is often characterized by fatty liver andinflammation. Alcoholic hepatitis can ultimately lead to cirrhosis ofthe liver (scarring) and hardening of the liver tissue. However,individuals that do not consume excessive amounts of alcohol can also befound to have liver disease complications. Non-alcoholic fatty liverdisease (NAFLD) is understood to encompass a variety of liver diseases,including steatosis (simple fatty liver), non-alcoholic steatohepatitis(NASH) and advanced scarring of the liver (cirrhosis). NASH hastraditionally been diagnosed by means of a liver biopsy to characterizethe liver histology, particularly with respect to the characteristics ofinflammation, fibrosis and steatosis (fat accumulation). NASH thengenerally refers to clinical findings based upon the liver biopsy of apatient with steatohepatitis, combined with the absence of significantalcohol consumption (Neuschwander-Tetri, B. A. and S. H. Caldwell (2003)Hepatology 37(5): 1202-1209).

In NASH, fat accumulation is seen in varying degrees of inflammation(hepatitis) and may lead to more serious conditions involving scarring(fibrosis). Patients having NASH are also often characterized byabnormal levels of liver enzymes, such as aspartate aminotransferase(AST) and alanine aminotransferase (ALT). Currently, there are fewtherapies to slow down or alter the course of further diseaseprogression in NASH. Therefore, there remains a need for effective NASHtreatments.

SUMMARY OF THE DISCLOSURE

The disclosure provides for a method for treating a fatty liver disorderin a subject in need thereof, comprising selecting a subject having orsuspected of having a fatty liver disease or disorder, wherein thesubject is non diabetic, pre-diabetic, or mildly diabetic; or has normalor substantially normal biliary tract function; and administering atherapeutically effective amount of a pharmaceutical compositioncomprising ethyl eicosapentanoate (EPA-E). In some cases EPA-E or EPApresent may be at least 40% by weight in total of the fatty acids andtheir derivatives.

In some embodiments, the present disclosure provides for a method fortreating a fatty liver disorder in a subject in need thereof, comprisingselecting a subject having or suspected of having a fatty liver diseaseor disorder, wherein the subject is non diabetic, pre-diabetic, ormildly diabetic; and administering a therapeutically effective amount ofa pharmaceutical composition comprising ethyl eicosapentanoate (EPA-E).In some cases the EPA-E present may be at least 40% by weight in totalof the fatty acids and their derivatives.

In some embodiments, the disclosure provides for a method for treating afatty liver disease or disorder in a subject in need thereof,comprising: selecting a subject having or suspected of having a fattyliver disease or disorder, wherein the subject's serum HbA1c level isequal to or less than 6.4% or the subject's fasting serum glucose levelis equal to or less than 125 mg/dl; and administering a therapeuticallyeffective amount of a pharmaceutical composition comprising ethyleicosapentanoate (EPA-E).

The disclosure also provides for a method for treating a fatty liverdisease or disorder in a subject in need thereof, comprising: selectinga subject having or suspected of having a fatty liver disease ordisorder, wherein the subject shows a normal or substantially normalbiliary function or a serum gamma glutamyl transferase (GGT) level isnormal or substantially normal, and administering a therapeuticallyeffective amount of a pharmaceutical composition comprising ethyleicosapentanoate (EPA-E).

In some embodiments, the disclosure provides for a method for treating afatty liver disease or disorder in a subject in need thereof,comprising: selecting a subject having or suspected of having a fattyliver disease or disorder, wherein the subject's serum HbA1c level isequal to or less than 6.4% or the subject's fasting serum glucose levelis equal to or less than 125 mg/dl; and administering a therapeuticallyeffective amount of a pharmaceutical composition comprising ethyleicosapentanoate (EPA-E); and administering in step b results in animprovement of serum EPA/AA ratio as compared to a baseline EPA/AA ratioequal to or greater than 0.1, 0.2, 0.3 or 0.4 in the subject. Thedisclosure also provides for a method for treating a fatty liver diseaseor disorder in a subject in need thereof, comprising: selecting asubject having or suspected of having a fatty liver disease or disorder,wherein the subject shows a normal or substantially normal biliaryfunction or a serum gamma glutamyl transferase (GGT) level equal to orless than 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50,55, 60 or 78 IU/L, or GGT level equal to or less than 30 or 85 IU/L formen and GGT level equal to or less than 24 or 55 IU/L for women andadministering a therapeutically effective amount of a pharmaceuticalcomposition comprising ethyl eicosapentanoate (EPA-E).

In some embodiments, the disclosure provides for a method for treating afatty liver disease or disorder in a subject in need thereof,comprising: selecting a subject having or suspected of having a fattyliver disease or disorder, wherein the subject's serum HbA1c level isequal to or less than 6.4% or the subject's fasting serum glucose levelis equal to or less than 125 mg/dl; wherein the subject is not treatedwith an anti-diabetic agent or has been previously treated with ananti-diabetic agent; and administering a therapeutically effectiveamount of a pharmaceutical composition comprising ethyl eicosapentanoate(EPA-E), wherein EPA-E present in the composition is at least 40% byweight in total of the fatty acids and their derivatives.

In some embodiments, the disclosure provides for a method for treating afatty liver disease or disorder in a subject in need thereof,comprising: selecting a subject having or suspected of having a fattyliver disease or disorder, wherein the subject shows a normal orsubstantially normal biliary function or a serum gamma glutamyltransferase (GGT) level equal to or less than 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 40, 45, 50, 55, 60 or 78 IU/L, or GGT levelequal to or less than 30 or 85 IU/L for men and GGT level equal to orless than 24 or 55 IU/L for women , (a) administering to a subject aneffective amount of a pharmaceutical composition comprising ethyleicosapentanoate (EPA-E); (b) improving the steatosis and lobularinflammation condition of said subject, and no worsening of saidfibrosis stage score; and (c) the subject exhibits the following changesin said at least one marker as compared to a baseline pretreatment levelof at least 1% reduction for ALT, AST, TG, TG/HDL ratio, Free fattyacid, AA, MUFA, Palmitoleic acid, Oleic acid, Oleic acid/Stearic acidratio, Palmitoleic acid/Palmitic acid ratio, Stearic acid/Palmitic acidratio, γ-linoleic acid/Linoleic acid ratio, Adrenic acid/AA ratio,Ferritin, Thioredoxin, TNFα, sTNF-R1, sTNF-R2, Hs-CRP, CTGF, sCD40,Leptin, complement factor D, CK18 fragment, serum HMGB1, Fas, Hyaluronicacid, Type IV collagen (7 s domain), procollagen III peptide or PAI-1;at least 5% increase for EPA or EPA/AA ratio; at least 1% increase forDPA, AA/Homo-γ-linoleic acid ratio or Serum adiponectin; no worsening ofALP, bilirubin, GGT, Albumin, HDL-C, LDL-C, TC, non-HDL-C, HOMA-IR,HbA1c, Glucose, Fasting plasma glucose, postprandial plasma glucose,OGTT, platelet count or BMI.

In some embodiments, the disclosure provides for a method for treating afatty liver disease or disorder in a subject in need thereof,comprising: selecting a subject having or suspected of having a fattyliver disease or disorder, wherein the subject's serum HbA1c level isequal to or less than 6.4% or the subject's fasting serum glucose levelis equal to or less than 125 mg/dl; administering a therapeuticallyeffective amount of a pharmaceutical composition comprising ethyleicosapentanoate (EPA-E) in combination with one or more anti-diabeticagents.

In some embodiments, the disclosure provides for a method for treating afatty liver disease or disorder in a subject in need thereof,comprising: selecting a subject where at least one criteria selectedfrom the group consisting of NAS score, steatosis score, lobularinflammation score, ballooning score and fibrosis stage is indicative offatty liver disease; and wherein the subject shows a serum gammaglutamyl transferase (GGT) level equal to or less than 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 55, 60 or 78 IU/L or GGTlevel equal to or less than 30 or 85 IU/L for men and GGT level equal toor less than 24 or 55 IU/L for women; and administering atherapeutically effective amount of a pharmaceutical compositioncomprising ethyl eicosapentanoate (EPA-E).

In some embodiments, the disclosure provides for a method for treating afatty liver disease or disorder in a subject in need thereof, comprisingselecting a subject having a: NAS score greater than or equal to 3; orsteatosis score equal to or greater than 1; or lobular inflammationscore equal to or greater than 1; or ballooning score equal to orgreater than 1; or fibrosis score equal to or greater than 1;administering a therapeutically effective amount of a pharmaceuticalcomposition comprising ethyl eicosapentanoate (EPA-E) to the subject;and wherein the subject's serum HbA1c level is equal to or less than6.4% or ii) the subject's fasting serum glucose level is equal to orless than 125 mg/dl.

In some embodiments, the present disclosure provides for a subject thatis non diabetic, pre-diabetic or mildly diabetic. In some aspects of thedisclosure, the subject receives no treatment for diabetes or noanti-diabetic agent. In some aspects of the disclosure, the subjectreceives at least one treatment for diabetes or at least oneanti-diabetic agent. In some aspects of the disclosure, the subjectreceives at least one treatment for diabetes or at least oneanti-diabetic agent is administered simultaneously with thepharmaceutical composition.

In some embodiments, the present disclosure provides for ananti-diabetic agent that is selected from the following group: PPARγagonists, biguanides, protein tyrosine phosphatase-1B (PTP-1B)inhibitors, meglitinides, a glucoside hydrolase inhibitors, insulinsecreatagogues, A2 antagonists, insulin and related compounds,non-thiazolidinediones, GSK 30/GSK 3 inhibitors, dipeptidyl peptidase IV(DP-IV) inhibitors, peptides, sulfonylureas, and nonsulfonylureasecretagogues.

In some embodiments, the present disclosure provides for a subject whoconsumes a diabetic diet or a western diet.

In some embodiments, the disclosure provides for a method for treating afatty liver disease or disorder in a subject in need thereof,comprising: selecting a subject having a: NAS score greater than orequal to 3; or steatosis score equal to or greater than 1; or lobularinflammation score equal to or greater than 1; or ballooning score equalto or greater than 1; or fibrosis score equal to or greater than 1;determining the subject's gamma glutamyl transferase (GGT) serum level;and administering a therapeutically effective amount of a pharmaceuticalcomposition comprising ethyl eicosapentanoate (EPA-E) to the subject, ifthe subject i) shows a serum gamma glutamyl transferase (GGT) levelequal to or less than 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 40,45, 50, 55, 60 or 78 IU/L or GGT level equal to or less than 30 or 85IU/L for men and GGT level equal to or less than 24 or 55 IU/L forwomen.

In some embodiments, the fatty liver disease or disorder is selectedfrom the group consisting of Non-Alcoholic Fatty Liver Disease (NAFLD)and Non-Alcoholic Steatohepatits (NASH).

In some embodiments, selecting a subject having or suspected of having afatty liver disease or disorder comprises selecting a subject having ascore selected from the group consisting of: a NAS score greater than orequal to 3, a steatosis score equal to or greater than 1, a lobularinflammation score equal to or greater than 1, a ballooning score equalto or greater than 1 and a fibrosis score equal to or greater than 1.

In some embodiments, the disclosure provides for selecting a subjecthaving or suspected of having a fatty liver disease or disorder, furthercomprising selecting a subject who is overweight.

In some embodiments, the disclosure provides for selecting a subjecthaving or suspected of having a fatty liver disease or disorder, furthercomprising selecting a subject with a body mass index (BMI) greater thanor equal to 25 kg/m².

In some embodiments, the disclosure provides for selecting a subjecthaving or suspected of having a fatty liver disorder, further comprisingselecting a subject with a familial history of fatty liver disease.

In some embodiments, the disclosure provides for selecting a subjecthaving or suspected of having a fatty liver disorder, further comprisingselecting a subject with a NAS score greater than or equal to 4.

In some embodiments, the disclosure provides for selecting a subjecthaving or suspected of having a fatty liver disorder comprises selectinga subject with a normal serum direct bilirubin level equal to or lessthan 0.4, 0.3, 0.2, 0.17 or 0.1.

In some embodiments, a subject is characterized by at least one criteriaselected from the group consisting of a baseline ALT value of about 10to about 300 IU/L; a baseline AST value of about 10 to about 250 IU/L; abaseline steatosis grade of about 2 to 3; and a baseline lobularinflammation grade of about 2 to 3.

In some embodiments, the present disclosure provides for fatty liverdisease characterized by the baseline pretreatment level in the subjectof at least one criteria selected from the group consisting of ALT in arange of 10 to 300 IU/L, AST in a range of 10 to 250 IU/L, HDL/C in arange of 25 to 55 mg/dl, LDL-C in a range of 100 to 200 mg/dl,triglycerides in a range of 100 to 1000 mg/dl, TC in a range of 170 to300 mg/dl, High TG and low HDL-C, TG/HDL-C ratio in a range of 3.75 to10, non-HDL-C in a range of 100 to 250 mg/dl, Free fatty acid in a rangeof 400 to 1000 μEq/L, HOMA-IR in a range of 1.5 to 5, HbA1c in a rangeof 5.7 to 10%, Fasting plasma glucose in a range of 100 to 200 mg/dl,impaired glucose tolerance and metabolic syndrome.

In some embodiments, the present disclosure provides for fatty liverdisease characterized by the baseline pretreatment level in the subjectof at least one criteria selected from the group consisting of low levelof EPA, DPA, DHA, EPA/AA, DHA/AA. DHA/DPA, AA/Homo-γ-linoleic acid: andhigh level of AA, MUFA, Palmitoleic acid, Oleic acid, Oleic acid/Stearicacid, Palmitoleic acid/Palmitic acid, γ-linoleic acid/Linoleic acid,Adrenic acid/AA compared to each average level in subjects with fattyliver disease.

In some embodiments, the present disclosure provides for administrationof EPA-E for about one year, and the subject exhibits at least oneimprovement selected from the group consisting of a reduced ALT value ascompared to said baseline ALT value; a reduced AST value as compared tosaid baseline AST value; a reduced steatosis grade as compared to saidbaseline steatosis grade; and a reduced lobular inflammation grade ascompared to said baseline lobular inflammation grade.

In some embodiments, the present disclosure provides for atherapeutically effective amount of EPA-E administered to the subject isan amount between about 1800 and about 2700 mg per day.

In some embodiments, the present disclosure provides for atherapeutically effective amount of EPA-E administered to the subject isat least 1800 mg per day.

In some embodiments, the present disclosure provides for atherapeutically effective amount of EPA-E administered to the subject isat least 2700 mg per day.

In some embodiments, the present disclosure provides for the subjectfurther characterized by having at least one condition selected from thegroup consisting of high triglycerides and low HDL-C, impaired glucosetolerance and metabolic syndrome.

In some embodiments, the present disclosure provides for reduced ALTvalue at least 5% lower than said baseline ALT value and/or said reducedAST value is at least 5% lower than said baseline AST value.

In some embodiments, the present disclosure provides for determining ina subject prior to treatment a baseline level in serum of at least onemember selected from the group consisting of ALT in a range of 10 to 300IU/L, AST in a range of 10 to 250 IU/L, HDL-C in a range of 25 to 55mg/dl, LDL-C in a range of 100 to 200 mg/dl, triglycerides in a range of100 to 1000 mg/dl, TC in a range of 170 to 300 mg/dl, High TG and lowHDL-C, TG/HDL-C ratio in a range of 3.75 to 10, non-HDL-C in a range of100 to 250 mg/dl, Free fatty acid in a range of 400 to 1000 μEq/L,HOMA-IR in a range of 1.5 to 5, HbA1c in a range of 5.7 to 10%, Fastingplasma glucose in a range of 100 to 200 mg/dl.

In some embodiments, the present disclosure provides for administrationof ethyl eicosapentanoate for at least 3 months, said subject exhibitsthe following changes in said at least one marker as compared to thebaseline level of at least 1% reduction for ALT, AST, TG, TG/HDL ratio,Free fatty acid, AA, MUFA, Palmitoleic acid, Oleic acid, Oleicacid/Stearic acid ratio, Palmitoleic acid/Palmitic acid ratio, Adrenicacid/AA ratio, Ferritin, Thioredoxin, TNFα, sTNF-R1, sTNF-R2, Hs-CRP,CRGF, sCD40, Leptin, complement factor D, CK18 fragment, serum HMGB1,Fas, Hyaluronic acid, Type IV collagen (7s domain), procollagen IIIpeptide or PAI-1; at least 5% increase for EPA or EPA/AA ratio; at least1% increase for DPA, AA/Homo-γ-linoleic acid ratio or Serum adiponectin;no worsening of ALP, bilirubin, GGT, Albumin, HDL-C, LDL-C, TC,non-HDL-C, HOMA-IR, HbA1c, Glucose, Fasting plasma glucose, postprandialplasma glucose, OGTT, platelet count or BMI.

In some embodiments, the present disclosure provides for improving thesteatosis and lobular inflammation condition of said subject, and noworsening of said fibrosis stage score.

In some embodiments, the present disclosure provides for improving theNAS score in said subject (i) to a composite score of 3 and no worseningof said fibrosis stage score, or (ii) by ≥2 across at least two of theNAS components and no worsening of said fibrosis stage score.

In some embodiments, the present disclosure provides for an improvementin serum EPA/AA ratio as compared to a baseline EPA/AA ratio.

In some embodiments, the present disclosure provides for improvement inserum EPA/AA ratio as compared to a baseline EPA/AA ratio equal to orgreater than 0.1, 0.2, 0.3 or 0.4.

In some embodiments, the present disclosure provides for thepharmaceutical composition administered to the subject 1 to 4 times perday.

In some embodiments, the present disclosure provides for the compositionpresent in one or more capsules.

In some embodiments, the present disclosure provides for the compositioncomprising a self-emulsifying composition comprising 50 to 95% byweight, a w3 polyunsaturated fatty acids and their pharmaceuticallyacceptable salts and esters.

In some embodiments, the present disclosure provides for the compositioncomprising a self-emulsifying composition comprising 50 to 95% byweight, EPA-E and its pharmaceutically acceptable salts and esters.

In some embodiments, the present disclosure provides for the compositioncomprising a self-emulsifying composition comprising at least 60%, byweight, EPA-E and its pharmaceutically acceptable salts and esters.

In some embodiments, the present disclosure provides for the compositioncomprising a self-emulsifying composition comprising at least 70%, byweight, EPA-E and its pharmaceutically acceptable salts and esters.

In some embodiments, the present disclosure provides for the compositioncomprising a self-emulsifying composition comprising at least 80%, byweight, EPA-E and its pharmaceutically acceptable salts and esters.

In some embodiments, the present disclosure provides for the compositioncomprising a self-emulsifying composition comprising at least 90%, byweight, EPA-E and its pharmaceutically acceptable salts and esters.

In some embodiments, the present disclosure provides for the compositioncomprising a self-emulsifying composition comprising at least 95%, byweight, EPA-E and its pharmaceutically acceptable salts and esters.

In some embodiments, the present disclosure provides for the compositioncomprising a self-emulsifying composition comprising at least 96%, byweight, EPA-E and its pharmaceutically acceptable salts and esters.

In some embodiments, the present disclosure provides for the compositioncomprising, 5 to 50% by weight, an emulsifier having a hydrophiliclipophilic balance of at least 10.

In some embodiments, the present disclosure provides for the compositioncomprising, 10 to 50% by weight, an emulsifier having a hydrophiliclipophilic balance of at least 10.

In some embodiments, the present disclosure provides for the compositioncomprising, 20 to 50% by weight, an emulsifier having a hydrophiliclipophilic balance of at least 10.

In some embodiments, the present disclosure provides for the compositioncomprising, 30 to 50% by weight, an emulsifier having a hydrophiliclipophilic balance of at least 10.

In some embodiments, the present disclosure provides for the compositioncomprising, 40 to 50% by weight, an emulsifier having a hydrophiliclipophilic balance of at least 10.

In some embodiments, the present disclosure provides for the compositioncomprising, ethanol content up to 4% by weight in relation to the totalcontent of the compound and the emulsifier.

In some embodiments, the present disclosure provides for the compositionnot containing ethanol.

In some embodiments, the present disclosure provides for the emulsifierat least one member selected from the group consisting ofpolyoxyethylene hydrogenated castor oil, polyoxyethylene sorbitan fattyacid ester, polyoxyethylene castor oil, polyethylene glycol fatty acidester, polyoxyethylene polyoxypropylene glycol, sucrose fatty acidester, and lecithin.

In some embodiments, the present disclosure provides for the emulsifieras at least one member selected from the group consisting ofpolyoxyethylene hydrogenated castor oil, polyoxyethylene sorbitan fattyacid ester, polyoxyethylene castor oil, and sucrose fatty acid ester.

In some embodiments, the present disclosure provides for thepolyoxyethylene hydrogenated castor oil as at least one member selectedfrom the group consisting of polyoxyethylene (20) hydrogenated castoroil, polyoxyethylene (40) hydrogenated castor oil, polyoxyethylene (50)hydrogenated castor oil, polyoxyethylene (60) hydrogenated castor oil,and polyoxyethylene (100) hydrogenated castor oil.

In some embodiments, the present disclosure provides for thepolyoxyethylene sorbitan fatty acid ester as at least one memberselected from the group consisting of polyoxyethylene sorbitanmonooleate, polyoxyethylene sorbitan tristearate, polyoxyethylenesorbitan monostearate, polyoxyethylene sorbitan monopalmitate, andpolyoxyethylene sorbitan monolaurate.

In some embodiments, the present disclosure provides for the sucrosefatty acid ester as at least one member selected from the groupconsisting of sucrose laurate, sucrose myristate, sucrose palmitate,sucrose stearate, and sucrose oleate.

In some embodiments, the present disclosure provides for the compositionas containing a lecithin where lecithin is at least one member selectedfrom the group consisting of soybean lecithin, enzymatically decomposedsoybean lecithin, hydrogenated soybean lecithin, and egg yolk lecithin.

In some embodiments, the present disclosure provides the composition ascontaining a polyhydric alcohol, where it may further comprise propyleneglycol or glycerin.

In some embodiments, the present disclosure provides the compositioncontains at least one member selected from the group consisting ofeicosapentaenoic acid, docosahexaenoic acid, and their pharmaceuticallyacceptable salts and esters.

In some embodiments, the present disclosure provides the compositioncontains less than 5% eicosapentaenoic acid, and their pharmaceuticallyacceptable salts and esters.

In some embodiments, the present disclosure provides the compositioncontains less than 5% docosahexaenoic acid, and their pharmaceuticallyacceptable salts and esters.

In some embodiments, the present disclosure provides the compositioncontains ethyl icosapentate and/or ethyl docosahexaenoate.

In some embodiments, the present disclosure provides the composition atotal content of the emulsifier having an HLB of at least 10 is 10 to100 parts by weight in relation to 100 parts by weight of the at leastone compound selected from the group consisting of w3 polyunsaturatedfatty acids and their pharmaceutically acceptable salts and esters.

In some embodiments, the present disclosure provides the composition atotal content of the emulsifier having an HLB of at least 10 is 10 to 50parts by weight in relation to 100 parts by weight of the at least onecompound selected from the group consisting of EPA-E and itspharmaceutically acceptable salts and esters.

In some embodiments, the present disclosure provides the subject's serumHbA1c level is equal to 6.4%, less than 6.4%, between 5.7% and 6.4%,equal to 5.6% or less than 5.6%

In some embodiments, the present disclosure provides the subject'sfasting serum glucose level is equal to 125 mg/dL, less than 125 mg/dL,between 100 mg/dL and 125 mg/dL, equal to 100 mg/dL or less than 100mg/dL.

In some embodiments, the present disclosure provides the subject's serumHbA1c level is measured using a technique selected from the followinggroup: high-performance liquid chromatography (HPLC); immunoassay;enzymatic assay; colorimetric assay; capillary electrophoresis andboronate affinity chromatography.

In some embodiments, the subject shows a serum gamma glutamyltransferase (GGT) level equal to or less than 24 IU/L.

In some embodiments the subject does not have a condition selected fromthe following group consisting of alcoholic liver injury, drug-inducedliver injury, chronic active hepatitis, cirrhosis, liver cancer, hepaticsteatosis and biliary tract disease.

In some embodiments, the present disclosure provides the subject'sglucose level is measured using methods selected from the following:FPG, RPG and OGTT.

In some embodiments the subject shows unobstructed or normal excretionof bile, does not suffer from injury in the liver, does not exhibitliver dysfunction, shows normal levels of direct bilirubin, does notpossess biliary tract disease or the subject possess biliary tractdisease in an early stage.

In some embodiments the subject consumes a diabetic diet or a westerndiet.

In some embodiments EPA-E is at least 40% by weight in total of thefatty acids and their derivatives in the composition.

In some embodiments, the pharmaceutical composition of the presentdisclosure may be any EPA-E containing compositions, includingcommercially available sources such as Lovaza™(Glaxo SmithKline, Fla.USA), Omacor™(Pronova Biopharma ASA, Oslo Norway), Lotriga™(TakedaPharmaceutical Co., Ltd., Osaka Japan) or Vascepa™(Amarin Pharma Inc.,NJ USA).

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent 4application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of a device of this disclosure are set forth withparticularity in the appended claims. A better understanding of thefeatures and advantages of this disclosure will be obtained by referenceto the following detailed description that sets forth illustrativeembodiments, in which the principles of a device of this disclosure areutilized, and the accompanying drawings.

FIG. 1 is a table representing the proportion of responders to EPA-Etreatment (NAS Score ≤3 or improvement of ≥2 across at least 2 of NAScomponents) in a group of non diabetic(including pre-diabetic) and milddiabetic patients.

FIG. 2 is a table representing further characterization of the patientsin FIG. 1. The table reflects the proportion of responders to EPA-Etreatment (NAS Score ≤3 or improvement of ≥2 across at least 2 of NAScomponents) in a sub-group of non diabetic(including pre-diabetic)patients with HbA1c levels =<6.4.

FIG. 3 is a table representing further characterization of the patientsin FIG. 1. The table reflects the proportion of responders to EPA-Etreatment (NAS Score ≤3 or improvement of ≥2 across at least 2 of NAScomponents) in a sub-group of non diabetic(including pre-diabetic) anddiabetic patients with fasting glucose levels=<125 mg/dL.

FIG. 4 is a table representing further characterization of the patientsin FIG. 1. The chart reflects the proportion of responders to EPA-Etreatment (NAS Score ≤3 or improvement of ≥2 across at least 2 of NAScomponents) in a sub-group of non diabetic(including pre-diabetic) anddiabetic patients with fasting glucose levels =<125 mg/dL and HbA1clevels =<6.4.

FIG. 5 is a table of possible criteria for the evaluation of NASH forbaseline scores before treatment, or after treatment.

FIG. 6 is a schematic of the experimental screen dosing at the end of aclinical trial study as described herein.

FIG. 7 is a table representing the proportion of responders to EPA-Etreatment (NAS Score ≤3 or improvement of ≥2 across at least 2 of NAScomponents) in a group of patients found to have γ-Glutamyl Transferase(GGT) levels ≤33 IU/L.

FIG. 8 is a table of γ-Glutamyl Transferase (GGT) levels in the patientsshown in FIG. 7 at the completion of the study.

FIG. 9 is a table representing further characterization of the patientsshown in FIG. 7. The table reflects the improvement in NAS score ofpatients with γ-Glutamyl Transferase (GGT) levels ≤33 IU/L.

FIG. 10 is a table representing further characterization of the patientsshown in FIG. 7. The table reflects the improvement of serum EPA/AAratio on Day 365 of patients with γ-Glutamyl Transferase (GGT) levels≤33 IU/L.

FIG. 11 is a table corresponding reference values for parameters ofliver function in patients with γ-Glutamyl Transferase (GGT) levels ≤33IU/L and patients with γ-Glutamyl Transferase (GGT) levels ≥33 IU/L.

FIG. 12 is a chart representing serum EPA/AA ratios for variousadministered dosages of EPA-E over time.

FIG. 13 is a chart representing serum ALP of patients with γ-GlutamylTransferase (GGT) levels ≤33 IU/L.

I. TERMINOLOGY

The terminology of the present disclosure is for the purpose ofdescribing particular embodiments only and is not intended to belimiting of compositions, methods and devices of this disclosure.

The terms “methods of treating” mean amelioration, prevention or relieffrom the symptoms and/or effects associated with NAFLD-associateddisorders. As used herein, reference to “treatment” of a patient isintended to include prophylaxis.

As used herein, a “therapeutically effective amount” of a drug orpharmaceutical composition or formulation, or agent, described herein isan amount of a drug or agent that, when administered to a subject with adisease or condition, will have the intended therapeutic effect, e.g.,alleviation, amelioration, palliation or elimination of one or moremanifestations of the disease or condition in the subject. The fulltherapeutic effect does not necessarily occur by administration of onedose and may occur only after administration of a series of doses. Thus,a therapeutically effective amount may be administered in one or moreadministrations.

A “subject” or “patient” is a mammal, preferably a human, but can alsobe an animal in need of veterinary treatment, e.g., companion animals(e.g., dogs, cats, and the like), farm animals (e.g., cows, sheep, pigs,horses, and the like) and laboratory animals (e.g., rats, mice, guineapigs, and the like).

The term “pre-diabetic” is the condition wherein an individual ispre-disposed to the development of type 2 diabetes. Pre-diabetes extendsthe definition of impaired glucose tolerance to include individuals witha fasting blood glucose within the high normal range 100 mg/dL (J. B.Meigs, et al. Diabetes 2003; 52:1475-1484) and fasting hyperinsulinemia(elevated plasma insulin concentration), fasting serum glucose levelsbetween 100 mg/dL and 125mg/dL, or HbA1c levels between 5.7% and 6.4%.The scientific and medical basis for identifying pre-diabetes as aserious health threat is laid out in a Position Statement entitled “ThePrevention or Delay of Type 2 Diabetes” issued jointly by the AmericanDiabetes Association and the National Institute of Diabetes andDigestive and Kidney Diseases (Diabetes Care 2002; 25:742-749).Pre-diabetic may be include in non-diabetic as provided by thisdisclosure.

The term “mildly-diabetic” is the condition wherein an individual inearly progression of the development of type 2 diabetes. Diabetesextends the definition of impaired glucose tolerance to includeindividuals with a fasting blood glucose within the high normal range≥126 mg/dL and/or HbA1c levels ≥6.5%. Mild-diabetes is diabetes whoreceives no treatment for diabetes, no ant-diabetic agents or only oneanti-diabetic agent.

The term “non-diabetic” is the condition wherein an individual does notpresent impaired glucose tolerance and includes individuals with afasting blood glucose within the normal range less than 100 mg/dL orHbA1c levels equal to or less than 5.6%.

The term “HbA1c” refers to the product of a non-enzymatic glycation ofthe haemoglobin B chain. Its determination is well known to one skilledin the art. In monitoring the treatment of diabetes mellitus the HbA1cvalue is of exceptional importance. As its production dependsessentially on the blood sugar level and the life of the erythrocytes,the HbA1c in the sense of a “blood sugar memory” reflects the averageblood sugar levels of the preceding 4-6 weeks.

The term “biliary tract function” is any anatomical, physiological orbiochemical function provided by the biliary tract in the body. Thebiliary tract is the common anatomical term for a duct that transportsbile, secreted by the liver, to the small intestine (duodenum). Asprovided by this disclosure, biliary traction function may also includesecretion of bile. Additionally, biliary tract function may also includethe excretion of bilirubin, a byproduct of red blood cells recycled bythe liver.

The term “normal” or “substantially normal” as applied to biliary tractfunction refers to unobstructed or otherwise functioning transport ofbile. Abnormal biliary tract function or biliary disease, usedinterchangeably herein, may include but is not limited to abnormal orelevated levels of markers associated with biliary disease, obstructionof the bial duct, decreased secretion of bile or bilirubin, failure tosecrete bile or bilirubin, abnormal pressure in the biliary duct,gallstones or cirrhosis of the liver as a result thereof.

The term “GGT” refers to γ-glutamyl transferase enzyme or gamma-glutamyltranspeptidase (also γ-glutamyltransferase, GGT, GGTP, gamma-GT). Thisenzyme catalyzes the transfer of gamma-glutamyl functional groups ofglutathione which is a strong anti-oxidant. It is found in many tissues,the most notable one being the liver, and has significance in medicineas a diagnostic marker. Other lines of evidence indicate that GGT canalso exert a prooxidant role, A digested product of glutathione“Cys-Gly”, in conjunction with metal ions, will produce active oxygen.Therefore, oxidation stress may increase when GGT level remains high.

The term “GGT test” refers to a common liver function test to test theactivity levels of GGT. Blood test results for GGT suggest that thenormal value is 8-78 IU/L (Merck Manual Appendix II, 2001, Merck Sharp &Dohme Corp., NJ USA), for men is 2-30 IU/L (Duh S H., LaboratoryReference Range Values, 2005, StedmansOnline, Lippincott Williams &Wilkins, PA USA) or 15-85 IU/L (General Laboratory Manual. Department ofPathology, Hackensack University Medical Centre. 2010. p. 117), whereasfor women it is 1-24 IU/L (Laboratory Reference Range Values) or 5-55IU/L (General Laboratory Manual. Department of Pathology, HackensackUniversity Medical Centre. 2010. p. 117).

The term “liver injury” or “liver disease” or “liver dysfunction” may beused interchangeably and refer to any injury of the liver, including butnot limited to hardening of the liver, scarring of the liver, decreasedor abnormal biliary tract function, abnormal liver enzyme activity,cirrhosis of the liver, abnormal physiology as determined by commondiagnostic methods include but not limited to ultrasound, orbiopsy/histopathology, necrosis of the liver and the like.

As used herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. Furthermore, to the extent that the terms “including”,“includes”, “having”, “has”, “with”, or variants thereof are used ineither the detailed description and/or the claims, such terms areintended to be inclusive in a manner similar to the term “comprising”.

Ranges can be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another embodiment includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the antecedent “about,” it willbe understood that the particular value forms another embodiment. Itwill be further understood that the endpoints of each of the ranges aresignificant both in relation to the other endpoint, and independently ofthe other endpoint. The term “about” as used herein refers to a rangethat is 15% plus or minus from a stated numerical value within thecontext of the particular usage. For example, about 10 would include arange from 8.5 to 11.5. The term “about” also accounts for typical erroror imprecision in measurement of values.

II. TREATMENT INDICATIONS

The methods and compositions of the present disclosure are useful forthe treatment of subjects having fatty liver related disorders and areknown, or suspected to be, non-diabetic, pre-diabetic, or mildlydiabetic, by administration of an effective amount of ethyleicosapentaenoic acid (EPA-E). The methods and compositions of thepresent disclosure are also useful for the treatment of subjects havingfatty liver related disorders and are known to have or suspected ofhaving normal or substantially normal biliary tract function, byadministration of an effective amount of ethyl eicosapentaenoic acid(EPA-E).

A. Fatty Liver Disorders

This disclosure provides compositions and methods for treating fattyliver disorders which may include but are not limited to non-alcoholicsteatohepatitis (NASH), non-alcoholic associated fatty liver disease,secondary NAFLD, steatosis, progressive fibrosis, liver failure andcirrhosis. As used herein, secondary NAFLD may refer to NAFLD or similarsymptoms that result from the use of one or more of the followingmedications: amiodarone, antiviral drugs such as nucleoside analogues,aspirin or NSAIDs, corticosteroids, methotrexate, nifedipine,perhexiline, tamoxifen, tetracycline, and valproic acid. The term fattyliver disorder, NASH, as described herein, may be referred to and usedinterchangeably as NASH herein.

B. Diabetes

This disclosure also provides compositions and methods for treating NASHsubjects who may also be or suspected to be non-diabetic, pre-diabetic,or mildly diabetic. Diabetes mellitus, commonly called diabetes, refersto a disease process derived from multiple causative factors andcharacterized by elevated levels of plasma glucose, referred to ashyperglycemia. There are two major forms of diabetes: Type 1 diabetes(also referred to as insulin-dependent diabetes or IDDM) and Type 2diabetes (also referred to as noninsulin dependent diabetes or NIDDM).Type 1 diabetes is the result of an absolute deficiency of insulin, thehormone that regulates glucose utilization. Type 1 diabetes has twoforms: Immune-Mediated Diabetes Mellitus, which results from a cellularmediated autoimmune destruction of the β cells of the pancreas; andIdiopathic Diabetes Mellitus, which refers to forms of the disease thathave no known etiologies. Type 2 diabetes is a disease characterized byinsulin resistance accompanied by relative, rather than absolute,insulin deficiency. Premature development of atherosclerosis andincreased rate of cardiovascular and peripheral vascular diseases arecharacteristic features of patients with diabetes.

Subjects referred herein as “diabetic” may have diabetes or associatedconditions. Diabetes, may include but is not limited to Type 1 diabetes,Type 2 diabetes, gestational diabetes mellitus (GDM), maturity onset ofdiabetes of the young (MODY), pancreatitis, polycystic ovarian disease,impaired glucose tolerance, insulin resistance, hyperglycemia,hyperinsulinemia, elevated blood levels of fatty acids or glycerol,obesity, Syndrome X, dysmetabolic syndrome and related diseases,diabetic complications (including retinopathy, neuropathy, nephropathy)and sexual dysfunction. The conditions, diseases, and maladiescollectively referenced to as “Syndrome X” or Dysmetabolic Syndrome (asdetailed in Johanson, J. Clin. Endocrinol. Metab., 1997, 82, 727-734,and other publications) include hyperglycemia and/or prediabetic insulinresistance syndrome, and is characterized by an initial insulinresistant state generating hyperinsulinemia, dyslipidemia, and impairedglucose tolerance, which can progress to Type II diabetes, characterizedby hyperglycemia, which can progress to diabetic complications or NAFLD.

C. Biliary Tract Function

This disclosure also provides compositions and methods for treating NASHsubjects who are known to have or suspected of having normal orsubstantially normal biliary tract function. As herein described,biliary tract function generally relates to any function of the biliaryof tract in the body, including anatomical, physiological andbiochemical function. The primary function of the biliary tract is thetransport of bile, secreted by the liver, to the small intestine. Asprovided by this disclosure, biliary tract function may also include thesecretion of bile.

Bile or gall, is a dark green, or yellowish fluid that aids in theprocess of the digestion of lipids in the small intestine. Bile isstored in the gallbladder and upon eating, is discharged into theduodenum. Bile may comprise water, bile salts, mucus and pigments, fatsand inorganic salts.

Bile may act a surfactant, aiding in emulsifying the fats in food. Bilesalts may be amphopathic, wherein salts contain both hydrophilic andhydrophobic elements. Bile may form micelles around hydrophobicmolecules, such as fats and fatty acids, wherein the hydrophobic sidesorient toward the hydrophobic molecule with the hydrophilic sides facingoutward. Bile is also used for the excretion of bilirubin, a byproductof red blood cells recycled by the liver. It is well known that bileenhances forming micelles and an absorption of fatty acids at smallintestine.

D. Evaluation criteria for subjects with NASH

Generally, any suitable method or combination of methods may be used inevaluating NASH in a subject. In terms of physical symptoms, NASH isgenerally asymptomatic until severe liver impairment occurs. Patientsmay generally feel well in the early stages and only begin to havesymptoms, such as fatigue, weight loss, and weakness once the disease ismore advanced or cirrhosis develops. The progression of NASH may takeyears, or even decades. The process can stop and, in some cases, reverseon its own without specific therapy. In some cases, NASH may slowlyworsen, causing scarring or “fibrosis” to appear and accumulate in theliver. As fibrosis worsens, cirrhosis develops; the liver becomesseriously scarred, hardened, and unable to function normally. Not everyperson with NASH develops cirrhosis, but once serious scarring orcirrhosis is present, few treatments can halt the progression. A personwith cirrhosis experiences fluid retention, muscle wasting, bleedingfrom the intestines, and liver failure. These physical symptoms in thelate stages of the disorder may be used to determine the presence orabsence of NASH in a subject.

i. Biopsy

Due to the asymptomatic nature of the disorder, particularly in earlystages of the disease, one or more technique and methods may be used toassess the absence or presence of NASH in a subject. In some cases, abiopsy is performed on the liver, whereby a needle is inserted throughthe skin to remove a small piece of the liver. NASH is diagnosed whenexamination of the tissue with a microscope shows fat along withinflammation and damage to liver cells. If the tissue shows fat withoutinflammation and damage, simple fatty liver or NAFLD is diagnosed. Insome cases, a biopsy may also indicate the presence or absence of scartissue that has developed in the liver. Currently, no blood tests orscans can reliably provide this information.

ii. Blood Tests and Biomarkers

While there are no single laboratory tests for NASH, various abnormallevels of liver enzymes, biomarkers and other biological bloodcomponents may be used in aiding diagnosis of the disorder. For example,in some cases, elevated serum aminotransferase may indicate NASH. Insome cases measuring the level of a plurality of suitable biomarkers ina sample derived from the subject may be used. In some cases such asadipocytokine, apoptosis markers, and/or cell death markers, may beanalyzed and compared to reference levels to aid in diagnosis of NASH.

In some cases subjects treated for NASH according to the presentdisclosure can also be evaluated for baseline scores of the followingcriteria before treatment, and evaluated after treatment for possiblechanges in those criteria. The evaluated criteria can comprise one ormore of the following criteria set forth in FIG. 5.

iii. NAS Score

In some cases, patients or subjects treated for NASH according to thepresent disclosure can also be evaluated using a combination ofstandardized histologic scores, as known in the art. Due to significantinter-observer variance in the clinical diagnosis of NASH, and theseverity of the disorder at given time, a composite score of histologicfeatures using a standardized NASH scoring system has been implementedand can be used to provide a measurement of NASH in a subject. Thiscombined measurement is known as the non-alcoholic fatty liver diseaseactivity score (NAS).

In some cases the NAS score may be determined for a subject prior toinitiation of treatment in order to provide a baseline level or scorefor the criteria as well as evaluation after the dosing regimen toevaluate any improvement in the criteria. In other cases, a NAS scoremay be determined for a subject undergoing treatment. In some cases, aNAS score and comparison of NAS scores over time may be used in assessdisease progression of NASH.

The non-alcoholic fatty liver disease activity score (NAS) is defined asthe unweighted sum of the values for steatosis (ranging from 0-3),lobular inflammation (ranging from 0-3) and ballooning (ranging from0-2), thereby providing a range of NAS score of from 0 to 8. (SeeKleinen et al., Design and Validation of a Histological Scoring Systemfor Nonalcoholic Fatty Liver Disease, Hepatology, Vol. 41, No. 6, 2005,pp. 1313-1321) Patients treated for NASH according to the presentdisclosure can show a NAS score prior to treatment of ≥4, with a minimumscore of 1 each for steatosis and lobular inflammation plus eitherballooning or at least 1 a sinusoidal fibrosis and a finding of possibleor definite steatohepatitis. After dosing/treatment, such as for oneyear, patients can show a composite NAS score of ≤3, ≤2 or ≤1, togetherwith no worsening in fibrosis. Alternatively, patients can show animprovement in NAS by a value of ≥2 across at least two of the NAScomponents, together with no worsening in fibrosis. Alternatively,patients can show an improvement in NAS score by ≥3, 4, 5, 6, 7 or 8.

iv. Steatosis

Steatosis is broadly understood to describe a process involving theabnormal accumulation of lipids within the liver, which inhibits normalliver function. Liver biopsy enables analysis and scoring of steatosisin a patient, with scores ranging from 0-3. Patients treated for NASHaccording to the present disclosure can have a steatosis score of 1, 2or 3, such as between about 2 and about 3. After treatment, it isdesired for patients to exhibit no worsening of steatosis, alternativelya reduction of at least 1 in the steatosis score, or a reduction of 2 or3 in the steatosis score. Steatosis is traditionally graded with a scoreof 1 indicating the presence of fat droplets in less than 33% ofhepatocytes, a score of 2 indicating fat droplets observed in 33-66% ofhepatocytes, and a score of 3 indicating observation of fat droplets ingreater than 66% of hepatocytes. (See Kleinen et al., Design andValidation of a Histological Scoring System for Nonalcoholic Fatty LiverDisease, Hepatology, Vol. 41, No. 6, 2005, pp. 1313-1321)

v. Lobular Inflammation

Lobular inflammation is also evaluated upon liver biopsy and scored withvalues of 0-3. (See Kleinen et al., Design and Validation of aHistological Scoring System for Nonalcoholic Fatty Liver Disease,Hepatology, Vol. 41, No. 6, 2005, pp. 1313-1321 Table 1) Patients to betreated for NASH can have lobular inflammation scores of 1, 2 or 3,alternatively ranging between 1 and 2 or 2 and 3. After treatment,patients can have a reduction in lobular inflammation score of at least1, alternatively a reduction of 2 or 3 in lobular inflammation score,and at least no worsening of the lobular inflammation score.

vi. Ballooning

Ballooning of hepatocytes is generally scored with values of 0-2, (SeeKleinen et al., Design and Validation of a Histological Scoring Systemfor Nonalcoholic Fatty Liver Disease, Hepatology, Vol. 41, No. 6, 2005,pp. 1313-1321 Table 1), and patients treated for NASH according to thepresent disclosure can have ballooning scores of 0-2, including specificvalues of 1 or 2, and alternatively a score ranging from 1 to 2. Aftertreatment, patients can show at least no worsening of the ballooningscore, alternatively a reduction of at least one value lower in theballooning score, and alternatively a reduction of two in the value ofthe ballooning score.

vii. Fibrosis Stage

Fibrosis is also evaluated upon liver biopsy and scored with values of0-4, the scores being defined as: 0 represents no fibrosis, 1 representsperisinusoidal or periportal fibrosis, la represents mild, zone 3,perisinusoidal fibrosis; lb represents moderate zone 3, perisinusoidalfibrosis; lc represents portal/periortal fibrosis; 2 representsperisinusoidal and portal/periportal fibrosis; 3 represents bridgingfibrosis; and 4 represents cirrhosis. (See Kleinen et al., Design andValidation of a Histological Scoring System for Nonalcoholic Fatty LiverDisease, Hepatology, Vol. 41, No. 6, 2005, pp. 1313-1321) Patientstreated according to the present disclosure can have a fibrosis stagescore of 0-3, including 0, 1, 1a, 1 b, 1 c, 2 or 3, and can have afibrosis stage score of at least la. After treatment, patients can havea fibrosis stage score that is at least no worse than the baselinescore, and alternatively can have a reduction in the fibrosis stagescore of at least one level, alternatively at least two or three levels.

E. Evaluation Criteria of Diabetes in NASH Subjects

As described herein, the methods and compositions of the presentdisclosure are useful for the treatment of subjects having fatty liverrelated disorders and are known, or suspected to be, non-diabetic,pre-diabetic, or mildly diabetic, by administration of an effectiveamount of ethyl eicosapentaenoic acid (EPA-E). The presence of absenceof non diabetes, pre-diabetes, or mild diabetes may be determined in asubject using any suitable methods known in the art. Generally,preferred tests for diabetes in NASH patients may be characterized intwo groups: serum glucose-based tests and glycated proteins. Serumglucose-based tests may include but are not limited to tests such asfasting plasma glucose (FPG), random plasma glucose (RPG), and the oralglucose tolerance test (OGTT). Tests of glycated proteins may includebut are not limited to tests that measure proteins such as HbA1c. Insome cases, one or more tests may be used to determine the presence orabsence of non diabetes, pre-diabetes, or mild diabetes. In some cases acombination of tests may be used to assess diabetes in NASH subjects.

i. Fasting Plasma Glucose (FPG)

The FPG test is a simple plasma glucose measurement obtained after atleast 8 hours of fasting (usually an overnight fast). This test may beused for screening and diagnosis of diabetes in NASH subjects due toease, expense and risk factors. FPG is the ADA test of choice fordiagnosis of both pre-diabetes and diabetes. When compared directly, FPGhas better intra-individual reproducibility than 2-hour post-load plasmaglucose, with intra-individual coefficients of variation of 6.4-11.4%for FPG versus 14.3-16.7% for 2-hour plasma glucose.

FPG may be a reliable predictor of diabetes complications at the currentthreshold for diagnosis, and studies examining FPG have underlined muchof the current knowledge about the pathology of diabetes. However, asknown in the art the threshold for pre-diabetes and its relationship tocomplications may vary across individuals or populations of individuals.General ranges for FPG for assessing non diabetes, pre-diabetes, or milddiabetes are described herein.

ii. Causal Plasma Glucose (RPG)

RPG (or “casual” plasma glucose) measurements may be easily obtainedfrom NASH subjects, do not require fasting, and are frequently includedin a basic metabolic panel ordered for other purposes. RPG tests may bedetermine diabetes, wherein a commonly accepted RPG threshold is ≥200mg/dl, along with symptoms of polyuria, polydipsia, and unexplainedweight loss to indicate a second test for confirmation of diagnosis. AnRPG of 140-199 mg/dl is suggestive of pre-diabetes. Based on diagnosisby OGTT, an RPG≥200 mg/dl is insensitive but has a specificityapproaching 100%.

Impairing the overall utility of the RPG as a testing tool is theabsence of data comparing it directly to rates of diabetes-specificcomplications. For this reason, use of the RPG test may be used forrapid, any-time testing with high specificity in symptomatic NASHsubjects.

iii. Oral Glucose Tolerance Test (OGTT)

Alternative tests for glucose also may include oral glucose tolerancetesting. In some cases it is the preferred test for diabetes diagnosis,.Regarding the diagnosis of diabetes, OGTT identifies about 2% moreindividuals than does FPG, OGTT has poor reproducibility compared toother glucose-based tests such as A1C.

HbA1c testing is known in the art as a standardized measure for diabetesdiagnosis that is now broadly used for both research and clinicalpurposes and may used to determine the presence of absence of nondiabetes, pre-diabetes, or mild diabetes in NASH subjects. Its majorpractical advantages are that it can be obtained in both fasting andnonfasting states, and it represents average glucose control over aperiod of months rather than a single point value. The ADA recommendsthis test a first-line test for screening and diagnosis. Atapproximately the same time, the International Expert Committee releasedthe formal recommendation of an HbA1c level >6.5% for diabetesdiagnosis.

HbA1c level may be measured using any suitable biochemical techniques.These may include but are not limited to high-performance liquidchromatography (HPLC); immunoassay; enzymatic assay; colorimetric assay;capillary electrophoresis and boronate affinity chromatography.

As with the glucose-based tests, there is no finite threshold of HbA1cat which normality ends and diabetes begins. The International ExpertCommittee has elected to recommend a cut point for diabetes diagnosisthat emphasizes specificity, commenting that this “balanced the stigmaand cost of mistakenly identifying individuals as diabetic against theminimal clinical consequences of delaying the diagnosis in someone withan HbA1c level <6.5%.

iv. General Thresholds for Evaluating Diabetes

The tests recommended for screening are the same as those for making thediagnosis, with the result that a positive screen is equivalent to adiagnosis of mild diabetes, pre-diabetes or diabetes. The term“pre-diabetes” has been assigned to those considered to be at higherrisk for developing diabetes. In some cases, pre-diabetes is diagnosedby having one or both of the following: 1) an FPG of 100-125 mg/dl,which is also referred to as impaired fasting glucose (IFG) or 2) a2-hour, 75-g OGTT, with 2-hour plasma glucose levels of 140-199 mg/dl,which is also described as IGT. To get a diagnosis of diabetes, patientsmust satisfy one of the following criteria: 1) symptoms of diabetes(polyuria, polydipsia, and unexplained weight loss) AND an RPG≥200mg/dl, 2) an FPG≥126 mg/dl, or 3) a 2-hour plasma glucose level ≥200mg/dl during a 75-g OGTT. Additional diagnostic criteria of an HbA1cresult ≥6.5% may also indicate pre-diabetes or mild diabetes. Subjectshaving mild diabetes may have levels at or about threshold levels asdescribed herein.

As already described herein, the term “pre-diabetic” in the presentdisclosure extends the definition of impaired glucose tolerance toinclude individuals with a fasting blood glucose within the high normalrange ≥100 mg/dL (J. B. Meigs, et al. Diabetes 2003; 52:1475-1484) andfasting hyperinsulinemia (elevated plasma insulin concentration), orHbA1c levels between 5.7% and 6.4%..

The term “mildly-diabetic” extends the definition of impaired glucosetolerance to include individuals with a fasting blood glucose within thehigh normal range 126 mg/dL and/or HbA1c levels ≥6.5% and who receivesno treatment for diabetes, no ant-diabetic agents or only oneanti-diabetic agent.

The term “non-diabetic” is the condition wherein an individual does notpresent impaired glucose tolerance and includes individuals with afasting blood glucose within the normal range less than 100 mg/dL orHbA1c levels less equal to or less than 5.6%.

In some instances, wherein a NASH subject may be pre-diabetic or mildlydiabetic, the NASH subject may undergo treatment or receive an antidiabetic agent. In some cases a NASH subject may have a western diet,which may comprise foods heavy in carbohydrates and fats. In othercases, a NASH subject may adopt a diabetic diet, which may comprise alower carbohydrate diet. In some cases, a NASH subject may receive oneor anti-diabetic agents alone or in combination with the pharmaceuticalcompositions of this disclosure. In some cases, the anti-diabetic may beadministered simultaneously as the pharmaceutical composition. In somecases, the pharmaceutical compositions are administered after a NASHsubject receives one or more anti-diabetic agents.

v. Anti-Diabetic Agents

In some cases as NASH a subject may be receive one or more anti diabeticagents which may include: PPARy agonists such as glitazones (e.g.,WAY-120,744, AD 5075, balaglitazone, ciglitazone, darglitazone(CP-86325, Pfizer), englitazone (CP-68722, Pfizer), isaglitazone(MIT/J&J), MCC-555 (Mitsibishi disclosed in U.S. Pat. No. 5,594,016),pioglitazone (such as such as Actos™ pioglitazone; Takeda),rosiglitazone (Avandia™; Smith Kline Beecham), rosiglitazone maleate,troglitazone (Rezulin®, disclosed in U.S. Pat. No. 4,572,912),rivoglitazone (CS-011, Sankyo), GL-262570 (Glaxo Welcome), BRL49653(disclosed in W098/05331), CLX-0921, 5-BTZD, GW-0207, LG-100641, JJT-501(JPNT/P&U), L-895645 (Merck), R 119702 (Sankyo/Pfizer), NN-2344 (Dr.Reddy/NN), YM-440 (Yamanouchi), LY-300512, LY-519818, R483 (Roche), T131(Tularik) and the like.

In some cases an anti diabetic agent may comprise biguanides such asmetformin hydrochloride (N,N-dimethylimidodicarbonimidic diamidehydrochloride, such as Glucophage™, Bristol-Myers Squibb); metforminhydrochloride with glyburide, such as GlucovanceTM, Bristol-MyersSquibb); buformin (Imidodicarbonimidic diamide, N-butyl-); etoformine(1-Butyl-2-ethylbiguanide, Schering A. G.); other metformin salt forms(including where the salt is chosen from the group of, acetate,benzoate, citrate, ftimarate, embonate, chlorophenoxyacetate, glycolate,palmoate, aspartate, methanesulphonate, maleate,parachlorophenoxyisobutyrate, formate, lactate, succinate, sulphate,tartrate, cyclohexanecarboxylate, hexanoate, octanoate, decanoate,hexadecanoate, octodecanoate, benzenesulphonate, trimethoxybenzoate,paratoluenesulphonate, adamantanecarboxylate, glycoxylate, glutamate,pyrrolidonecarboxylate, naphthalenesulphonate, 1-glucosephosphate,nitrate, sulphite, dithionate and phosphate), and phenformin; bile acidsequestrants that include, but are not limited to, cholestyramine (i.e.,QUESTRAN®, QUESTRAN LIGHT®, CHOLYBAR®, CA registry no. 11041-12-6),colesevelam (i.e., WELCHOL®, CA registry nos. 182815-43-6 and182815-44-7), ursodeoxycholic acid (i.e. CA registry no. 128-13-2),colestipol (i.e., COLESTID®, CA registry nos. 50925-79-6 and37296-80-3), sevelamer, dialkylaminoalkyl derivatives of a cross-linkeddextran, LOCHOLEST®, DEAE-Sephadex (SECHOLEX®, POLIDEXIDEL®), watersoluble derivatives such as 3,3-ioene, N-(cycloalkyl)alkylamines andpoliglusam, insoluble quaternized polystyrenes, saponins and mixturesthereof, those bile acid sequestrants disclosed in WO97/11345,WO98/57652, U.S. Pat. No. 3,692,895, and U.S. Pat. No. 5,703,188,including pharmaceutically acceptable salts or mixtures thereof Suitableinorganic cholesterol sequestrants include bismuth salicylate plusmontmorillonite clay, aluminum hydroxide and calcium carbonate antacids.

In some cases an anti diabetic agent may comprise protein tyrosinephosphatase-1B (PTP-1B) inhibitors, such as A-401,674, KR 61639,OC-060062, OC-83839, OC-297962, MC52445, MC52453, ISIS 113715, and thosedisclosed in WO99/585521, WO99/58518, WO99/58522, WO99/61435,WO03/032916, WO03/032982, WO03/041729, WO03/055883, WO02/26707,WO02/26743, JP2002114768, and pharmaceutically acceptable salts andesters thereof; sulfonylureas such as acetohexamide (e.g., Dymelor, EliLilly), carbutamide, chlorpropamide (e.g., Diabinese®, Pfizer),gliamilide (Pfizer), gliclazide (e.g., Diamcron, Servier Canada Inc),glimepiride (e.g., disclosed in U.S. Pat. No. 4,379,785, such asAmarylTM, Aventis), glipentide, glipizide (e.g., Glucotrol or GlucotrolXL Extended Release, Pfizer), gliquidone, glisolamide,glyburide/glibenclamide (e.g., Micronase or Glynase Prestab, Pharmacia &Upjohn and Diabeta, Aventis), tolazamide (e.g., Tolinase), andtolbutamide (e.g., Orinase), and pharmaceutically acceptable salts andesters thereof.

In some cases an anti diabetic agent may comprise meglitinides such asrepaglinide (e.g., Pranidin®, Novo Nordisk), KAD1229 (PF/Kissei), andnateglinide (e.g., Starlix®, Novartis), and pharmaceutically acceptablesalts and esters thereof;

In some cases an anti diabetic agent may comprise a glucoside hydrolaseinhibitors (or glucoside inhibitors) such as acarbose (e.g., Precose™,Bayer disclosed in U.S. Pat. No. 4,904,769), miglitol (such as GlysetTM,Pharmacia & Upjohn disclosed in U.S. Pat. No. 4639436), camiglibose(Methyl6-deoxy-6-[(2R,3R,4R,5S)-3,4,5-trihydroxy-2-(hydroxymethyl)piperidino]-alpha-D-glucopyranoside,Marion Merrell Dow), voglibose (Takeda), adiposine, emiglitate,pradimicin-Q, salbostatin, CKD-711, MDL-25,637, MDL-73,945, and MOR 14,and the like.

In some cases an anti diabetic agent may comprise insulin secreatagoguessuch as linogliride, A-4166, forskilin, dibutyrl cAMP,isobutylmethylxanthine (IBMX), and pharmaceutically acceptable salts andesters thereof, fatty acid oxidation inhibitors, such as clomoxir, andetomoxir, and pharmaceutically acceptable salts and esters thereof.

In some cases an anti diabetic agent may comprise A2 antagonists, suchas midaglizole, isaglidole, deriglidole, idazoxan, earoxan, andfluparoxan, and pharmaceutically acceptable salts and esters thereof.

In some cases an anti diabetic agent may comprise insulin and relatedcompounds (e.g., insulin mimetics) such as biota, LP-100, novarapid,insulin detemir, insulin lispro, insulin glargine, insulin zincsuspension (lente and ultralente), Lys-Pro insulin, GLP-1 (1-36) amide,GLP-1 (73-7) (insulintropin, disclosed in U.S. Pat. No. 5,614,492),LY-315902 (Lilly), GLP-1 (7-36)-NH2), AL-401 (Autoimmune), certaincompositions as disclosed in U.S. Pat. No. 4,579,730, U.S. Pat. No.4,849,405, U.S. Pat. No. 4,963,526, U.S. Pat. No. 5,642,868, U.S. Pat.No. 5,763,396, U.S. Pat. No. 5,824,638, U.S. Pat. No. 5,843,866, U.S.Pat. No. 6,153,632, U.S. Pat. No. 6,191,105, and WO 85/05029, andprimate, rodent, or rabbit insulin including biologically activevariants thereof including allelic variants, human insulin available inrecombinant form (sources of human insulin include pharmaceuticallyacceptable and sterile formulations such as those available from EliLilly (Indianapolis, Ind. 46285) as HumulinTM (human insulin rDNAorigin), also see the the Physician's Desk Reference, 55.sup.th Ed.(2001) Medical Economics, Thomson Healthcare (disclosing other suitablehuman insulins).

In some cases an anti diabetic agent may comprise non-thiazolidinedionessuch as JT-501 and farglitazar (GW-2570/GI-262579), and pharmaceuticallyacceptable salts and esters thereof; PPARα/γ dual agonists such asAR-H039242 (Aztrazeneca), GW-409544 (Glaxo-Wellcome), BVT-142, CLX-0940,GW-1536, GW-1929, GW-2433, KRP-297 (Kyorin Merck; 5-[(2,4-Dioxothiazolidinyl)methyl]methoxy-N-[[4-(trifluoromethyl)phenyl]methyl]benzamide),L-796449, LR-90, MK-0767 (Merck/Kyorin/Banyu), SB 219994, muraglitazar(BMS), tesaglitzar (Astrazeneca), reglitazar (JTT-501) and the like.

In some cases an anti diabetic agent may comprise GSK 30/GSK 3inhibitors such as4-[2-(2-bromophenyl)-4-(4-fluorophenyl-1H-imidazol-5-yl]pyridine and thelike.

In some cases an anti diabetic agent may comprise dipeptidyl peptidaseIV (DPP-IV) inhibitors, or peptides including amlintide and Symlin®(pramlintide acetate).

F. Evaluation Criteria of Biliary Function in NASH Subjects

As described herein, the methods and compositions of the presentdisclosure are useful for the treatment of subjects having fatty liverrelated disorders and are known, or suspected to have normal orsubstantially normal biliary tract function, by administration of aneffective amount of ethyl eicosapentaenoic acid (EPA-E). The presence ofabsence of normal or substantially normal biliary tract function may bedetermined in a subject using any suitable methods known in the art.Generally, preferred tests for biliary tract function in NASH patientsmay be characterized in two groups: physiological based tests andbiochemical based tests. Physiological based tests may include but arenot limited to abdominal ultrasound, abdominal CT scan, endoscopicretrograde cholangiopancreatography (ECRP), Percutaneous transhepaticcholangiogram (PTCA) or Magnetic resonance cholangiopancreatography(MRCP). Biochemical based tests may include but are not limited to GGTtests, liver function tests, bilirubin tests, alkaline phosphatase (ALP)tests, liver enzyme tests, amylase blood test, lipase blood test,prothrombin time, and measurement of urine bilirubin. In some cases, oneor more tests may be used to characterize biliary function. In somecases a combination of tests may be used to assess biliary function inNASH subjects.

i. Physiological Tests

Generally, the physiological tests, examples of which are providedherein, provide a visualization of the biliary duct, which may aid inthe diagnosis of an obstruction. For example, if gallstones are present,these may be result in obstruction or partial obstruction of the biliaryduct and the presence of gallstones may be visualized. In some casesvisualization is achieved with X-rays (PTCA or abdominal CT), magneticresonance (MRCRP) or ultrasound. In some cases, a direct obstruction maynot be visualized. In some cases, these methods may indicate thenarrowing of the biliary duct, or secondary effect of an obstruction.These may also aid in evaluating biliary function in a NASH subject.

ii. GGT test

The GGT test is a common liver function enzyme test that measures theactivity of the enzyme GGT. Blood test results for GGT suggest that thenormal value is 8-78 IU/L (Merck Manual Appendix II), for men is 2-30IU/L (Laboratory Reference Range Values) or 15-85 IU/L (GeneralLaboratory Manual. Department of Pathology, Hackensack UniversityMedical Centre. 2010. p. 117), whereas for women it is 1-24 IU/L(Laboratory Reference Range Values) or 5-55 IU/L (General LaboratoryManual. Department of Pathology, Hackensack University Medical Centre.2010. p. 117).

In some cases, elevated serum GGT activity may be indicative of diseasesof the liver, biliary system, and pancreas. In this respect, it issimilar to alkaline phosphatase (ALP) tests, as described herein indetecting disease of the biliary tract. Both ALP and GGT may be used toas biochemical indicator of potential liver disease, although GGT testsgenerally provide increased sensitivity. Slightly elevated serum GGT mayalso been found to correlate with cardiovascular diseases in NASHsubjects. GGT may accumulate in atherosclerotic plaques, and maycirculate in blood in the form of distinct protein aggregates, some ofwhich may indicate specific pathologies such as metabolic syndrome,alcohol addiction and chronic liver disease. High body mass index (BMI)may be associated with type 2 diabetes subjects with high serum GGT.Tests for GGT levels may be used alone or in combination with othertests to assess biliary tract function in NASH subjects.

iii. ALP Test

The ALP test is a common liver function enzyme test that measures theactivity of the enzyme ALP. The normal range is 36-92 IU/L (Merck ManualAppendix II) or 20 to 140 IU/L(General Laboratory Manual. Department ofPathology, Hackensack University Medical Centre. 2010. p. 117) High ALPlevels can show that the bile ducts are obstructed. Levels aresignificantly higher in children and pregnant women.

iv. Additional Biochemical Based Tests

Generally, any suitable biochemical tests may be used in the assessmentof biliary function of NASH subjects. In some cases, tests, as part of astandard liver function panel, may be used to assess biliary function.In some cases, bilirubin tests may be useful to assess biliary function.One or more tests may be used in combination tests as provided herein inthe assessment of biliary function.

iv. General Thresholds for Evaluating Biliary Function

The tests recommended for screening are the same as those for making thediagnosis, with the result that a positive screen is equivalent to adiagnosis of normal or substantially normal biliary function. In thiscase, normal or substantially normal may also comprise a subject beingat risk for biliary disease. In some cases, biliary disease is diagnosedby having one or both of the following: 1) GGT equal to or more than 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 55, 60 or 78IU/L or GGT level equal to or less than 30 or 85 IU/L for men and GGTlevel equal to or less than 24 or 55 IU/L for women 2) an ALP test equalto or more than 92 or 140 IU/L. To get a diagnosis of abnormal biliaryfunction, patients must satisfy one of the following criteria: 1)symptoms of biliary disease (i.e. narrowing or obstruction of the bialduct, weight loss, pain in the abdomen,) AND abnormal levels of GGT orALP. Additional diagnostic criteria of one or more of these indicatorsmay also indicate abnormal biliary function. In some cases, biliarydisease is diagnosed by having serum direct bilirubin equal to or morethan 0.3 or 0.4. Subjects having early stages of biliary disease mayhave levels at or about threshold levels as described herein.

III. PHARMACEUTICAL COMPOSITION A. EPA-E Compositions

Eicosapentaenoic acid (EPA) is a known omega-3 polyunsaturated,long-chain fatty acid. Omega-3 fatty acids are known as components ofoils, such as fish oil. A variety of commercial products are promoted ascontaining omega-3 fatty acids, or their esters, derivatives, conjugatesand the like. Eicosapentaenoic acid (EPA) is also known as its ethylester form, ethyl eicosapentanoate (EPA-E). According to the presentdisclosure, EPA-E can be administered in a composition. EPA-E content inthe total fatty acid of the compositions of the present disclosure arenot particularly limited as long as the composition contains EPA-E asits effective component and intended effects of the present disclosureare attained, high purity EPA-E is preferably used.

The present disclosure may be a self-emulsifying composition comprising50 to 95% by weight in total of EPA-E of preferably 60% by weight ormore, more preferably 90% by weight or more, and still more preferably96.5% by weight or more in total of the fatty acids and theirderivatives. In some cases, the pharmaceutical composition may compriseat least about 40%, 46%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, and 96.5% byweight of EPA-E in total of the fatty acids and their derivatives. Insome cases, the pharmaceutical composition may comprise at most about40%, 46%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, and 96.5% weight of EPA-Ein total of the fatty acids and derivatives. EPA-E can be administeredto patients in a highly purified form, including the product known asEpadel® (Mochida Pharmaceutical Co., Ltd., Tokyo Japan) andpharmaceutically acceptable salts and esters thereof.

The EPA-E used in the present disclosure may be a synthetic,semi-synthetic, natural EPA-E, or a natural oil containing such EPA-E.Examples of the natural EPA-E include an extract from a natural oilcontaining an EPA-E, a crudely purified natural oil containing an EPA-E,and a highly purified natural oil containing an EPA-E produced by amethod known in the art. Exemplary semi-synthetic EPA-E include EPA-Eproduced by a microorganism or the like and the EPA-E or the naturalEPA-E which have been subjected to a chemical treatment such asesterification or ester exchange.

B. Formulation

In some embodiments, omega-3 fatty acids are formulated as aself-emulsifying composition. Self-emulsifying composition of thepresent disclosure may preferably have at least one of the effectsincluding excellent self-emulsifying property, excellent dispersibilityin the composition, excellent emulsion stability, excellent storagestability, excellent absorption property, and in particular, excellentabsorption property and rate under fasting, and excellent convenience orcompliance for the patients so that the composition can exhibitpharmacological effect of the EPA-E. In some cases, EPA-E may becombined with 5 to 50% by weight of an emulsifier having an HLB of atleast 10.

In some cases, the pharmaceutical composition may comprise at leastabout 5%, 10%, 20%, 30%, 40%, and 50% emulsifier. In some cases, thepharmaceutical composition may comprise at most about 5%, 10%, 20%, 30%,40%, and 50% emulsifier. The self-emulsifying composition is free fromethanol or the ethanol content is low. The present disclosure alsoprovides a drug of such self-emulsifying composition, its productionmethod, and the method of its use.

Examples of the pharmaceutically acceptable salts of the EPA-E includeinorganic salts such as sodium salts and potassium salts, organic saltssuch as benzylamine salts and diethylamine salts, salts with basic aminoacids such as arginine salts and lysine salts, and exemplary estersinclude alkyl esters such as ethyl ester, and esters such as mono-, di-and TG. Preferable examples include ethyl ester and TG ester, and themore preferred is ethyl ester. More specifically, preferable examplesinclude EPA-E, TG ester of EPA-E, and the like.

The EPA-E used for the starting material of the self-emulsifyingcomposition of the present disclosure is not particularly limited forits purity. The purity is typically such that content of the EPA-E intotal of the fatty acids and their derivatives in the composition of thepresent disclosure could be preferably at least 40%, 50% or 60% byweight, more preferably at least 70% by weight, still more preferably atleast 80% by weight, still more preferably at least 90% by weight, andmost preferably at least 96.5% by weight. The EPA-E containing EPA-E andDHA-E at a high purity, for example, the one with the content of(EPA-E+DHA-E) in relation to the EPA-E of at least 50% by weight intotal of the fatty acids and their derivatives is preferable, and thecontent is more preferably at least 60% by weight in total of the fattyacids and their derivatives, still more preferably at least 90% byweight in total of the fatty acids and their derivatives, and mostpreferably at least 98% by weight in total of the fatty acids and theirderivatives. In other words, the composition of the present disclosurepreferably has a high purity of EPA-E in the total fatty acid.

For example, when EPA-E and DHA-E are used, compositional ratio ofEPA-E/DHA-E and content of (EPA-E+DHA-E) in relation to total fatty acidare not particularly limited as long as the purity of EPA in thecomposition of the present disclosure is in the range as describedabove. However, the compositional ratio of the EPA-E/DHA-E is preferablyat least 0.8, more preferably at least 1.0, and most preferably at least1.2.

The composition of the present disclosure may also contain apolyunsaturated fatty acid other than the EPA-E such as linoleic acid, ylinolenic acid, or dihomo-γ-linolenic acid or the pharmaceuticallyacceptable salt or ester thereof. However, content of arachidonic acidin the total of the fatty acids and their derivatives is preferably low,more preferably less than 2% by weight, still more preferably less than1% by weight, and most preferably, the composition is substantially freefrom the arachidonic acid.

Compared to the fish oil or the fish oil concentrate, the EPA-E used inthe composition or therapeutic agent of the present disclosure containsimpurities such as saturated fatty acids and arachidonic acid which areunfavorable for cardiovascular events at a lower content, and thisenables realization of the intended action without causing the problemsof excessive nutrition or vitamin A intake. When the EPA-E in the formof ester is used, a sufficiently stable composition can be obtained byadding a commonly used antioxidant since the ester form has higheroxidation stability than the fish oils which are mainly TG form.

Purified fish oils may also be used for the EPA-E, and use ofmonoglyceride, diglyceride, and TG derivatives and combinations thereofof the EPA-E are also preferable embodiments. Various productscontaining the EPA-E are commercially available, for example, IncromegaF2250, F2628, E2251, F2573, TG2162, TG2779, TG2928, TG3525, and E5015(Croda International PLC, Yorkshire, England), and EPAX6000FA,EPAX5000TG, EPAX4510TG, EPAX2050TG, EPAX7010EE, K85TG, K85EE, and K8OEE(Pronova Biopharma, Lysaker, Norway). These products may be purchasedand used for the composition of the present disclosure.

In the present disclosure, the “polyoxyethylene hydrogenated castor oil”is a compound prepared by addition polymerization of ethylene oxide tothe hydrogenated castor oil which is castor oil having hydrogen addedthereto, Various compounds with different average degree ofpolymerization of ethylene oxide are commercially available, andexamples include polyoxyethylene (20) hydrogenated castor oil (NTKKOLHCO-20, Nikko Chemicals Co., Ltd.), polyoxyethylene (40) hydrogenatedcastor oil (NIKKOL HCO-40, Nikko Chemicals Co., Ltd.), polyoxyethylene(50) hydrogenated castor oil (NIKKOL HCO-50, Nikko Chemicals Co., Ltd.),polyoxyethylene (60) hydrogenated castor oil (NIKKOL HCO-60, NikkoChemicals Co., Ltd.), and polyoxyethylene (100) hydrogenated castor oil(NIKKOL HCO-100, Nikko Chemicals Co., Ltd.), and the preferred ispolyoxyethylene (60) hydrogenated castor oil. These may be used alone orin combination of two or more. In the present disclosure, the“polyoxyethylene hydrogenated castor oil” includes all of such compoundsunless otherwise noted.

In the present disclosure, the “polyoxyethylene sorbitan fatty acidester” is polyoxyethylene ether of a fatty acid ester wherein a part ofthe hydroxy groups of anhydrous sorbitol have been esterified with afatty acid. Various compounds with different esterified fatty acid arecommercially available, and examples include polyoxyethylene (20)sorbitan monolaurate (NIKKOL TL-10, Nikko Chemicals Co., Ltd.),polyoxyethylene (20) sorbitan monopalmitate (NIKKOL TP-10V, NikkoChemicals Co., Ltd.), polyoxyethylene (20) sorbitan monostearate (NIKKOLTS-10V, Nikko Chemicals Co., Ltd.), polyoxyethylene (20) sorbitantristearate (NIKKOL TS-30V, Nikko Chemicals Co., Ltd.), polyoxyethylene(20) sorbitan monoisostearate (NIKKOL TI-10V, Nikko Chemicals Co.,Ltd.), polyoxyethylene (20) sorbitan monooleate (NIKKOL TO-10V, NikkoChemicals Co., Ltd.), and polyoxyethylene (20) sorbitan trioleate(NIKKOL TO-30V, Nikko Chemicals Co., Ltd.), and the preferred arepolyoxyethylene (20) sorbitan monolaurate, polyoxyethylene (20) sorbitanmonopalmitate, polyoxyethylene (20) sorbitan monostearate, andpolyoxyethylene (20) sorbitan monooleate, and the more preferred arepolyoxyethylene (20) sorbitan monooleate. These may be used alone or incombination of two or more, In the present disclosure, the“polyoxyethylene sorbitan fatty acid ester” includes all of suchcompounds unless otherwise noted.

In the present disclosure, the “polyoxyethylene castor oil” is acompound prepared by addition polymerization of ethylene oxide to castoroil. Various compounds having different average ethylene oxide molenumber are commercially available, and examples include NIKKOL CO-3 withan average ethylene oxide mole number of 3 (Nikko Chemicals Co., Ltd.),NIKKOL CO-10 with an average ethylene oxide mole number of 10 (NikkoChemicals Co., Ltd.), EMALEX C-20 with an average ethylene oxide molenumber of 20 (Nippon Emulsion Co., Ltd.), EMALEX C-30 with an averageethylene oxide mole number of 30 (Nippon Emulsion Co., Ltd.), EMALEXC-40 with an average ethylene oxide mole number of 40 (Nippon EmulsionCo., Ltd.), and EMALEX C-50 with an average ethylene oxide mole numberof 50 (Nippon Emulsion Co., Ltd.). These may be used alone or incombination of two or more. In the present disclosure, the“polyoxyethylene castor oil” includes all of such compounds unlessotherwise noted.

In the present disclosure, the “polyethylene glycol fatty acid ester” isa fatty acid ester of a polyethylene glycol which is a fatty acidpolymerized with ethylene oxide. Various compounds with differentesterified fatty acid are commercially available, and examples includepolyethylene glycol monolaurate (NIKKOL MYL-10, Nikko Chemicals Co.,Ltd.), polyethylene glycol monostearate (NIKKOL MYS-10V, MYS-25V,MYS-40V, NYS-45V, and MYS-55V, Nikko Chemicals Co., Ltd.), polyethyleneglycol monooleate (NIKKOL MYO-6 and MYO-10, Nikko Chemicals Co., Ltd.),polyethylene glycol distearate (NIKKOL CDS-6000P, Nikko Chemicals Co.,Ltd.), and polyethylene glycol diisostearate (NIKKOL CDIS-400, NikkoChemicals Co., Ltd.). These may be used alone or in combination of twoor more. In the present disclosure, the “polyethylene glycol fatty acidester” includes all of such compounds unless otherwise noted.

In the present disclosure, the “polyoxyethylene polyoxypropylene glycol”is a compound prepared by addition polymerization of ethylene oxide tothe polypropylene glycol which is a polymerized propylene oxide. Variouscompounds having different average degree of polymerization of thepropylene oxide and the ethylene oxide are commercially available, andexamples include polyoxyethylene (3) polyoxypropylene (17) glycol (AdekaPluronic L-31, ADEKA), polyoxyethylene (20) polyoxypropylene (20) glycol(Adeka Pluronic L-44, ADEKA), polyoxyethylene (42) polyoxypropylene (67)glycol (Adeka Pluronic P-123, ADEKA), polyoxyethylene (54)polyoxypropylene (39) glycol (Newdet PE-85, Sanyo Chemical Industries,Ltd.), polyoxyethylene (105) polyoxypropylene (5) glycol (PEP101, SanyoChemical Industries, Ltd.), polyoxyethylene (120) polyoxypropylene (40)glycol (Adeka Pluronic F-87, ADEKA), polyoxyethylene (160)polyoxypropylene (30) glycol (Adeka Pluronic F-68, ADEKA),polyoxyethylene (196) polyoxypropylene (67) glycol (Lutrol F127, BASFJapan), and polyoxyethylene (200) polyoxypropylene (70) glycol, and thepreferred is polyoxyethylene (105) polyoxypropylene (5) glycol. Thesemay be used alone or in combination of two or more. In the presentdisclosure, the “polyoxyethylene polyoxypropylene glycol” includes allof such compounds unless otherwise noted.

In the present disclosure, the “sucrose fatty acid ester” is an ester ofsugar and a fatty acid. Various compounds with different types of theesterified fatty acids and degree of esterification are commerciallyavailable, and examples include Surfhope SE PHARMA J-1216 containing 95%of lauric acid in the fatty acid (Mitsubishi-Kagaku Foods Corporation),Surfhope SE PHARMA J-1416 containing 95% of myristic acid in the fattyacid (Mitsubishi-Kagaku Foods Corporation), Surfhope SE PHARMA J-1615and J-1616 containing 80% of palmitic acid in the fatty acid,(Mitsubishi-Kagaku Foods Corporation), J-1811, J-1815, and J-1816containing 70% of stearic acid in the fatty acid (Mitsubishi-Kagakuroods Corporation), and Surfhope SE PHARMA J-1715 containing 70% ofoleic acid in the fatty acid, which may be used alone or in combinationof two or more. The “sucrose fatty acid ester” used in the presentdisclosure include all of such compounds.

The emulsifier added to a self-emulsifying composition of the presentdisclosure may have an HLB of at least 10, preferably at least 11, andmore preferably at least 12.

Total content of the emulsifier having an HLB of at least 10 in aself-emulsifying composition of the present disclosure is notparticularly limited as long as it is at least 10 parts by weight inrelation to 100 parts by weight of the EPA-E. The content is typically10 to 100 parts by weight, preferably 10 to 80 parts by weight, and morepreferably 10 to 50 parts by weight in relation to 100 parts by weightof the EPA-E.

In the present disclosure, the “lecithin” is a type ofglycerophospholipid, and examples include soybean lecithin,enzymatically decomposed soybean lecithin, hydrogenated soybeanlecithin, egg yolk lecithin, hydrogenated phospholipid, phospholipidfrom milk, lysolecithin, phosphatidyl choline, and phosphatidyl serine.The preferred are soybean lecithin, enzymatically decomposed soybeanlecithin, hydrogenated soybean lecithin, and egg yolk lecithin, and themore preferred are soybean lecithin. These may be used alone or incombination of two or more. In the present disclosure, the “lecithin”includes all of such compounds unless otherwise noted.

Commercially available products include purified soybean lecithin(Nisshin Oilio), purified egg yolk lecithin (Asahi Kasei PharmaCorporation), and egg yolk lecithin PL-100M (Kewpie Corporation), anduse of such product is also possible.

In the present disclosure, the “polyhydric alcohol” is a polyol compoundhaving the structure of a straight chain or cyclic aliphatic hydrocarbonwherein two or more carbon atoms are each substituted with one hydroxygroup. Exemplary such polyhydric alcohols include divalent alcohols suchas ethyleneglycol, propylene glycol, trimethylene glycol, 1,2-butyleneglycol, tetramethylene glycol, 1,3-butylene glycol, 2,3-butylene glycol,and pentamethylene glycol; trivalent alcohols such as glycerin,trimethylolpropane, and 1,2,6-hexane triol, and polyhydric alcoholpolymers such as diethylene glycol, dipropylene glycol, triethyleneglycol, polyethylene glycol, polypropylene glycol, and polyglycerin, andthe preferred are propylene glycol or glycerin. In the presentdisclosure, the “polyhydric alcohol” includes all of such compoundsunless otherwise noted.

Total amount of the lecithin and/or the polyhydric alcohol added in theself-emulsifying composition of the present disclosure is notparticularly limited. However, the total amount of the lecithin and/orthe polyhydric alcohol is typically 0 to 50 parts by weight, preferably3 to 40 parts by weight, and more preferably 5 to 30 parts by weight inrelation to 100 parts by weight of the EPA-E.

The ethanol in the self-emulsifying composition of the presentdisclosure is preferably used at an amount not causing quality change inthe course of capsulation, distribution, or storage, at an amount notcausing change in the content of the capsule, and at an amount notexceeding the established upper limit of the daily dose as a drug. Theethanol content is typically up to 10% by weight, preferably up to 4% byweight, more preferably up to 1% by weight, more preferably up to 0.5%by weight, more preferably up to 0.2% by weight, still more preferablyup to 0.1% by weight, and most preferably 0% by weight (no ethanoladdition).

Preferable ethanol concentration can be adequately determined inconsideration of the EPA-E concentration in the self-emulsifyingcomposition and the daily dose. When the self-emulsifying composition ofthe present disclosure is orally administered at a daily dose in termsof the EPA-E of 1800 mg, and for example, the preparation contains 75%by weight of the EPA-E, the maximum daily dose of 3.26 mg described in“Dictionary of Drug Additives (in Japanese)” will not be exceeded whenthe ethanol content is not more than 0.135% by weight.

The preferable embodiment of the self-emulsifying composition of thepresent disclosure containing such EPA-E and an emulsifier is acombination of EPA-E and/or DHA-E with at least one emulsifier selectedfrom the group consisting of polyoxyethylene hydrogenated castor oil,polyoxyethylene sorbitan fatty acid ester, polyoxyethylene castor oil,sucrose fatty acid ester, and lecithin. When the self-emulsifyingcomposition of the present disclosure is used as a food such as specialpurpose food, functional health food, and health food, the preferred isthe combination of EPA-E and/or DHA-E with a sucrose fatty acid esterand/or a lecithin which has good results as a food additive. When asucrose fatty acid ester is used, the preferable amount is 1% by weightto 20% by weight, more preferably 4% by weight to 20% by weight, andmost preferably 4% by weight to 10% by weight in the self-emulsifyingcomposition. The most preferable embodiments are a combination of EPA-Eand polyoxyethylene (50) hydrogenated castor oil or polyoxyethylene (60)hydrogenated castor oil; a combination of EPA-E and polyoxyethylene (20)sorbitan monooleate; a combination of EPA-E and polyoxyethylene castoroil; and a combination of EPA-E and sucrose fatty acid ester J-1216 orJ-1816.

Also preferred is the further combination with a lecithin such assoybean lecithin and/or a polyhydric alcohol such as propylene glycol.

When the emulsifier is at least one member selected from the groupconsisting of polyoxyethylene hydrogenated castor oil, polyoxyethylenesorbitan fatty acid ester, and polyoxyethylene castor oil, thepolyhydric alcohol is preferably a dihydric alcohol, and use ofpropylene glycol is more preferable. When the emulsifier is a sucrosefatty acid ester, the polyhydric alcohol is preferably a trihydricalcohol, and use of glycerin is more preferable.

Preferably, the composition and therapeutic agent of the presentdisclosure is substantially free from water. The “substantially freefrom water” means that the water content is up to 10% by weight,preferably up to 5% by weight, and even more preferably up to 3% byweight.

The self-emulsifying composition of the present disclosure may alsocontain additives such as an emulsion aid, stabilizer, antiseptic,surfactant, and antioxidant. Exemplary emulsion aids include fatty acidscontaining 12 to 22 carbon atoms such as stearic acid, oleic acid,linoleic acid, palmitic acid, linolenic acid, and myristic acid andtheir salts. Exemplary stabilizers include phosphatidic acid, ascorbicacid, glycerin, and cetanols, and exemplary antiseptics include ethylparaoxybenzoate and propyl paraoxybenzoate. Exemplary surfactantsinclude sucrose fatty acid esters, sorbitan fatty acid esters, glycerinfatty acid esters, polyglycerin fatty acid esters, polyoxyethylenesorbitan fatty acid esters, polyoxyethylene alkyl ethers,polyoxyethylene fatty acid esters, polyoxyethylene alkyl phenyl ethers,and polyoxyethylene polyoxypropylene alkyl ethers having an HLB of lessthan 10. Exemplary antioxidants include oil-soluble antioxidants such asbutylated hydroxy toluene, butylated hydroxy anisole, propyl gallate,propyl gallate, pharmaceutically acceptable quinone, astaxanthin, andα-tocopherol.

In addition, an adequate carrier or mediator, a colorant, a flavor, andoptionally, a vegetable oil or an additive such as non-toxic organicsolvent or non-toxic solubilizing agent (for example glycerin),emulsifier, suspending agent (for example, Tween 80 and gum arabicsolution), isotonic agent, pH adjusting agent, stabilizer, corrective,flavoring agent, preservative, antioxidant, or absorption promotercommonly used in the art may be adequately combined to prepare anappropriate pharmaceutical preparation.

More specifically, since the EPA-E is highly unsaturated, effectiveamount of an oil-soluble antioxidant, for example, at least one memberselected from butylated hydroxytoluene, butylated hydroxyanisole, propylgallate, propyl gallate, pharmaceutically acceptable quinone,astaxanthin, and α-tocopherol is preferably incorporated in thecomposition. Storage temperature is preferably room temperature, andfrozen storage is preferably avoided since the freezing may result inthe loss of self-emulsifying property, dispersibility in thecomposition, or emulsion stability.

The self-emulsifying composition of the present disclosure can beproduced by mixing the EPA-E, the emulsifier having an HLB of at least10, and the optionally added components such as lecithin, polyhydricalcohol, and antioxidant with optional heating to dissolve thecomponents.

In some cases, the pharmaceutical composition may comprise additionalelements which may include but are not limited to antihypertensives suchas angiotensin II receptor blockers such as irbesartan, olmesartanmedoxomil, candesartan cilexetil, telmisartan, valsartan, and losartanpotassium; angiotensin-converting enzyme inhibitors such as alacepril,imidapril hydrochloride, enalapril maleate, captopril, quinaprilhydrochloride, cilazapril hydrate, temocapril hydrochloride, delaprilhydrochloride, trandolapril, benazepril hydrochloride, perindopril, andlisinopril hydrate; calcium antagonists such as azelnidipine, amlodipinebesylate, aranidipine, efonidipine hydrochloride, cilnidipine,nicardipine hydrochloride, nifedipine, nimodipine, nitrendipine,nilvadipine, barnidipine hydrochloride, felodipine, benidipine, andmanidipine; α receptor blocker such as tolazoline, and phentolamine; βreceptor blockers such as atenolol, metoprolol, acebutolol, propranolol,pindolol, carvedilol, and labetalol hydrochloride; a receptors stimulantsuch as clonidine and methyldopa; and diuretics such as eplerenone,hydrochlorothiazide, and furosemide.

In some cases, the pharmaceutical composition may comprise additionalelements which may include but are not limited to antidiabetics asdescribed herein.

In some cases, the pharmaceutical composition may comprise additionalelements which may include but are not limited to vitamins such asascorbic acid (vitamin C), tocopherol (vitamin E), and tocopherolnicotinate, and N-acetylcysteine, probucol.

In some cases, the pharmaceutical composition may comprise additionalelements which may include but are not limited to blood flow improvingagents such as cilostazol, ticlopidine hydrochloride, alprostadil,limaprost, beraprost sodium, sarpogrelate hydrochloride, argatroban,naftidrofuryl, isoxsuprine hydrochloride, batroxobin, dihydroergotoxinemesilate, tolazoline hydrochloride, hepronicate, and shimotsu-toextract.

In some cases, the pharmaceutical composition may comprise additionalelements which may include but are not limited to bile acid derivativessuch as ursodeoxycholic acid, chenodeoxycholic acid, bile powder,deoxycholic acid, cholic acid, bile extract, bear bile, oriental bezoar,and dehydrocholic acid. Preferable examples also include biotin (vitaminB7), cyanocobalamin (vitamin B12), pantothenic acid (vitamin B5), folicacid (vitamin B9), thiamine (vitamin B1), vitamin A, vitamin D, vitaminK, tyrosine, pyridoxine (vitamin B6), branched chain amino acids such asleucine, isoleucine, and valine, calcium, iron, zinc, copper, andmagnesium. Other examples include components used in designated healthfoods and functional nutritional foods such as soy protein, chitosan,low molecular weight sodium alginate, dietary fiber from psyllium seedcoat, soy peptide with bound phospholipids, phytosterol ester, plantstanol ester, diacylglycerol, globin digest, and tea catechin.

C. Administration and Dosage

Compositions comprising EPA-E useful for the disclosure includecommercially available compositions of EPA-E, such as Epadel®, Lovaza™,Omacor™, Lotriga™ or Vascepa™ noted above. Compositions comprising EPA-Emay be administered in tablet, capsule, powder or any other solid oraldosage form, as a liquid, as a soft gel capsule or other capsule form,or other appropriate and convenient dosage forms for administration to apatient in need thereof. Compositions can also include pharmaceuticallyacceptable excipients known to those of ordinary skill in the artincluding surfactants, oils, co-solvents or combinations of suchexcipients, together with stabilizers, emulsifiers, preservatives,solubilizers and/or other non-active pharmaceutical ingredients known tothose of skill in the art relative to the preparation of pharmaceuticalcompositions.

The dose and dosage period of the EPA-E used in the composition of thepresent disclosure is a dose and period sufficient for realizing theintended action, which may be adequately adjusted depending on theadministration route, frequency of administration per day, seriousnessof the symptoms, body weight, age, and other factors.

A composition of the present disclosure may be administered to thepatient orally, endorectally, or transvaginally. However, oraladministration is preferable in the case of the patient who can take thedrug orally, and the composition may be administered in the form of ajelly preparation in the case of patients undergoing dialysis orpatients with aphagia by jelling the composition with gelatin or thelike.

Doses of the aforementioned compositions as the active ingredient can besuitably decided depending on the purpose of administration, i.e.,therapeutic or preventive treatment, nature of a disease to be treatedor prevented, conditions, body weight, age, sexuality and the like of apatient. The practically desirable method and sequence foradministration varies depending on the purpose of administration, i. e.,therapeutic or preventive treatment, nature of a disease to be treatedor prevented, conditions, body weight, age, sexuality and the like of apatient. The optimum method and sequence for administration of thecompounds described in detail herein under preset given conditions maybe suitably selected by those skilled in the art with the aid of theroutine technique and the information contained in the presentspecification and field of invention.

In the case of oral administration, the composition may be administeredat a dose in terms of the EPA-E of 0.1 to 5 g/day, preferably 0.2 to 3g/day, more preferably 0.4 to 2.0 g/day, and most preferably 0.6 to 1.0g/day in 1 to 3 divided doses. However, the entire dose may beadministered at once or in several divided doses. While meal affectsabsorption of the EPA-E, and the administration of the EPA-E ispreferably conducted during the meal or after the meal, and morepreferably immediately after the meal (within 30 minutes after themeal), the self-emulsifying composition of the present disclosure hasexcellent absorption under fasting, and therefore, it exhibits theintended effects even when administered at a timing other than during,after, or immediately after the meal, for example, before or immediatelybefore the meal or before going to the bed; to patients with reducedabsorption ability of the intestinal tract (for example, elderly,patients of intestinal disease, patients after intestinal surgery,terminal cancer patients, or patients taking a lipase inhibitor); orused at a reduced dose.

The compositions of EPA-E are administered according to the disclosureto a subject or patient to provide the patient with a dosage of about0.3-10 g per day of EPA-E, alternatively 0.5-8g per day, alternatively0.6-6 g per day, alternatively 1-4g per day, alternatively 0.9-3.6 g perday or specifically about 1800 mg per day or about 2700 mg per day ofEPA-E.

The composition to be administered can contain other fatty acids,especially any omega-3 unsaturated fatty acid, especially DHA-E. Theratio of EPA-E/DHA-E in the composition, the content of EPA-E and DHA-Ein the total fatty acids and administration amount of EPA-E and DHA-Eare not limited but the ratio is preferably 0.8 or more, more preferably1.0 or more, still more preferably 1.2 or more. The composition ispreferably highly purified; for example, the proportion of EPA-E+DHA-Ein the fatty acids and their derivatives is preferably 40% by weight ormore, more preferably 80% by weight or more, and still more preferably90% by weight or more. The daily amount in terms of EPA-E+DHA-E istypically 0.3 to 10.0 g/day, preferably 0.5 to 6.0 g/day, and still morepreferably 1.0 to 4.0 g/day. The low content of other long chainsaturated fatty acids is preferred, and among the long chain unsaturatedfatty acids, the content of omega-6 fatty acids, and in particular, thecontent of arachidonic acid in total of the fatty acids and theirderivatives is preferably as low as less than 2% by weight, and morepreferably less than 1% by weight. For example, soft capsule (Lovaza™,Omacor™ and Lotriga™) containing about 46% by weight of EPA-E and about38% by weight of DHA-E is commercially available in the U.S. and othercountries as a therapeutic agent for hypertriglyceridemia and softcapsule (VascepaTM) containing at least 96% by weight of EPA-E iscommercially available in the U.S as a therapeutic agent forhypertriglyceridemia.

Patients treated for NASH can be administered EPA-E according to thedisclosure for 3, 6 or 9 months, or for 1 year, 2 years or more and canbe administered EPA-E in one, two or three dosage per day, or othermultiple doses per day including 1 to about 10, 1 to 8, 1 to 6, 1 to 4or 1 to 2 dosage units per day as appropriate for patient therapy. Theterm “dose unit” and “dosage unit” herein refer to a portion of apharmaceutical composition that contains an amount of EPA-E for a singleadministration to a subject.

When orally administered at such dose, the administration period may beadequately determined depending on the target disease and degree ofsymptoms. For example, in the case of administration for NASH, theadministration period is not particularly limited as long asimprovements of biochemical markers related to NASH, improvement in thepathological conditions or therapeutic effects, and suppression of theprogress in metabolic syndrome, cardio or cerebrovascular event, orulcer and gangrene of extremities and peripheries are realized. However,administration period is determined to realize the improvements in theconcentration of plasma lipid marker (total cholesterol (hereinafterabbreviated as Cho), TG, postprandial TG, low-density lipoprotein Cho,high-density lipoprotein Cho, very-low-density lipoprotein Cho,non-high-density lipoprotein Cho, intermediate-density lipoprotein Cho,very-high-density lipoprotein Cho, free fatty acid, phospholipid,chylomicron, ApoB, lipoprotein (a), remnant-like lipoprotein Cho, smalldense low-density lipoprotein Cho, etc.), increase in the skintemperature of extremities and peripheries which can be measured bythermography or the like, increase in the walking distance, increase inthe serum CPK or other test value, and improvement of various symptomssuch as numbness, coldness, ache, pain at rest, itching, cyanosis,flare, chilblain, neck stiffness, anemia, poor complexion, itching, andcrawling. The amelioration or therapeutic effects may be monitored byother biochemical, pathological, or symptomatic parameters related toNASH. The administration is preferably continued as long as abnormalityis observed in biochemical index such as serum lipid concentration orpathology. In addition, the composition may be administered everyalternate day or 2 or 3 days in a week, or as the case may be, a drugwithdrawal period of about 1 day to 3 month, and more preferably about 1week to 1 month may be included.

If indicated by the physician, oral administration may be started at adose lower than the recommended daily EPA-E dose at the first day, andthen, the dose may be gradually increased to the maximum daily dose asthe maintenance dose. The dose may be reduced depending on theconditions of the patient. Lower daily dose is preferable in view ofreducing the side effects, and administration of once or twice a day ispreferable in view of the drug compliance.

The method of the present invention may administer a therapeuticallyeffective amount of a pharmaceutical composition comprising EPA-E incombination with a second effective component. The second effectivecomponent may be adequately determined depending on the target diseaseand the seriousness of the symptom. However, the second effectivecomponent is preferably a component that does not adversely affect theeffects of EPA-E, and examples include therapeutic agent forhyperlipidemia, antihypertensives, antidiabetics, antioxidants, bloodflow improving agents, and bile acid derivatives.

Of the preferable examples of the second effective component, exemplarytherapeutic agents for hyperlipidemia includepolyenephosphatidylcholine, unsaponifiable soybean oil (soy sterol),gamma-oryzanol, riboflavin butyrate, dextran sulfate sodium sulfur 18,pantethine, and elastase; statins such as pravastatin, simvastatin,atorvastatin, fluvastatin, pitavastatin, rosuvastatin, and cerivastatin;fibrates such as simfibrate, clofibrate, clinofibrate, bezafibrate, andfenofibrate; lipolytic enzyme inhibitors such as orlistat andcetilistat; resins such as colestyramine and colestimide; and ezetimibe.

Exemplary antihypertensives include angiotensin II receptor blockerssuch as irbesartan, olmesartan medoxomil, candesartan cilexetil,telmisartan, valsartan, and losartan potassium; angiotensin-convertingenzyme inhibitors such as alacepril, imidapril hydrochloride, enalaprilmaleate, captopril, quinapril hydrochloride, cilazapril hydrate,temocapril hydrochloride, delapril hydrochloride, trandolapril,benazepril hydrochloride, perindopril, and lisinopril hydrate; calciumantagonists such as azelnidipine, amlodipine besylate, aranidipine,efonidipine hydrochloride, cilnidipine, nicardipine hydrochloride,nifedipine, nimodipine, nitrendipine, nilvadipine, barnidipinehydrochloride, felodipine, benidipine, and manidipine; [alpha] receptorblocker such as tolazoline, and phentolamine; [beta] receptor blockerssuch as atenolol, metoprolol, acebutolol, propranolol, pindolol,carvedilol, and labetalol hydrochloride; a receptors stimulant such asclonidine and methyldopa; and diuretics such as eplerenone,hydrochlorothiazide, and furosemide.

Exemplary antidiabetics include [alpha]-glucosidase inhibitors such asacarbose, voglibose, and miglitol; sulfonyl urea hypoglycemics such asgliclazide, glibenclamide, glimepiride, and tolbutamide; fast-actinginsulin secretagogues such as nateglinide and mitiglinide; biguanidehypoglycemics such as metformin hydrochloride and buforminhydrochloride; dipeptidyl phosphatase 4 inhibitors such as sitagliptin,vildagliptin, alogliptin, linagliptin and saxagliptin; thiazolidinereagents such as pioglitazone hydrochloride and rosiglitazone maleate;and glucagon-like peptide 1 derivative reagents such as exenatide,lixisenatide and liraglutide.

Exemplary antioxidants include vitamins such as ascorbic acid (vitaminC), tocopherol (vitamin E), and tocopherol nicotinate, andN-acetylcysteine, probucol.

Exemplary blood flow improving agents include cilostazol, ticlopidinehydrochloride, alprostadil, limaprost, beraprost sodium, sarpogrelatehydrochloride, argatroban, naftidrofuryl, isoxsuprine hydrochloride,batroxobin, dihydroergotoxine mesilate, tolazoline hydrochloride,hepronicate, and shimotsu-to extract.

Exemplary bile acid derivatives include ursodeoxycholic acid,chenodeoxycholic acid, bile powder, deoxycholic acid, cholic acid, bileextract, bear bile, oriental bezoar, and dehydrocholic acid. Preferableexamples also include biotin (vitamin B7), cyanocobalamin (vitamin B12),pantothenic acid (vitamin B5), folic acid (vitamin B9), thiamine(vitamin B1), vitamin A, vitamin D, vitamin K, tyrosine, pyridoxine(vitamin B6), branched chain amino acids such as leucine, isoleucine,and valine, calcium, iron, zinc, copper, and magnesium. Other examplesinclude components used in designated health foods and functionalnutritional foods such as soy protein, chitosan, low molecular weightsodium alginate, dietary fiber from psyllium seed coat, soy peptide withbound phospholipids, phytosterol ester, plant stanol ester,diacylglycerol, globin digest, and tea catechin.

D. Treatment Effects

The compositions of the present disclosure can be used as a therapeuticagent for various diseases of animals, and in particular, mammals, forexample, therapeutic agent for NASH and related disorders. Thecompositions of the present disclosure are particularly expected toexhibit amelioration or therapeutic effects in NASH subjects withoutdiabetes, or with pre-diabetes or mild diabetes, or without biliarytract disease, or in the early stages of the disease. NASH subjects mayimprove or maintain a variety of symptoms, which may include but are notlimited to, increase in blood lipid, expression of insulin resistance,increase in blood pressure, abnormal liver function tests, abnormalliver enzyme activity, elevated glucose levels, liver dysfunction,hyperlipidemia, or any abnormal criteria as found in FIG. 5. In somecases, the pharmaceutical composition may aid in maintaining a function,level or activity present in a subject.

In some cases, the compositions may lower fasting glucose levels bymethods as provided by the disclosure. In some cases, the compositionmay lower glucose levels as determined to be above normal, as would befound in pre-diabetic and mildly diabetic subjects. In some cases, thecomposition may lower high glucose levels in subjects not considered tohave diabetes. In some cases, the pharmaceutical composition may aid inmaintaining a glucose level present in a subject.

In some cases, the composition may lower GGT levels lower than thethresholds as provided by the disclosure. In some cases, the compositionmay lower GGT levels as determined to be above normal, as would be foundin NASH subjects having biliary disease or be at risk for the disease.In some cases, the composition may lower GGT levels in subjects notconsidered to have biliary disease. In some cases, the pharmaceuticalcomposition may aid in maintaining a GGT level present in a subject.

The compositions of the present disclosure can reduce burden of thepatients by reducing the dose and daily frequency of the administration,and hence, by improving the drug compliance. This also results in thehigher effects of amelioration or treatment.

IV. EXAMPLES Example 1 Pre-Clinical Experience

In animal and in vitro model studies, EPA-E (ethylall-cis-5,8,11,14,17-eicosapentaenoate) has been shown to lower lipidsin rats, hamsters and rabbits; have anti-aggregation effects onplatelets from rats, rabbits and humans; and to preserve the elasticityof arteries in rabbits. In other studies, polyunsaturated fatty acids(PUFAs) have been shown to ameliorate hepatic steatosis in ob/ob micethrough down-regulation of hepatic nuclear sterol regulatory elementbinding protein-1c (SREBP-1c). In a similar manner, EPA-E followingrepeat oral administration at >0.1 mg/g suppressed fat accumulation in amouse diet-induced hepatic steatosis model by suppressing hepaticSREBP-1c levels as well as monounsaturated fatty acid (MUFA) synthesisby stearoyl-Coenzyme A desaturase 1 (SCD1). In a galactosamine-inducedsteatohepatitis mouse model, EPA-E after oral administration at 1000mg/kg retarded progression of steatohepatitis by suppressingtriglyceride (TG) accumulation. EPA-E following repeat oraladministration at 1000 mg/kg inhibited fibrosis by suppressinginflammation and oxidative stress in a methionine-choline deficient dietrat model of nonalcoholic steatohepatitis.

In safety pharmacology studies, at oral doses up to 3000 mg/kg, EPA-Ehad no effect on the central nervous, autonomic nervous, respiratory andcardiovascular systems except for a reduction in gastric fluid levels inpylorus-ligated rats after a 3000 mg/kg oral dose. The effects of themetabolites and impurities of EPA-E as well as oxidized EPA-E on theabove systems were not marked and do not appear to significantlycontribute to general pharmacological effects of EPA-E.

Pharmacokinetics and Product Metabolism in Animals Summary

EPADEL is a product containing ethylall-cis-5,8,11,14,17-eicosapentaenoate (EPA-E), one of the n-3 essentialfatty acids. The preclinical absorption, distribution, metabolism andexcretion (ADME) characteristics of EPA-E have been determined primarilyin rats.

Oral administration of a single dose of radioactive ¹⁴C-EPA-E at thedose of 30-1000 mg/kg in rats showed that EPA-E was well-absorbed,mainly through the lymphatic route. After administration of ¹⁴C-EPA-Einto the ligated intestine of rats, the residual radioactivity in theintestine at 24 hours was only 4.6% of the administered dose.Radioactivity transferred to the plasma and lymph was mainly detected inthe triglyceride (TG) fraction in the early phase after dosing, whiledistribution to the free fatty acid (FFA) fraction was slight at alltimepoints. The radioactivity was widely distributed in the bodytissues; relatively high levels were observed especially in the brownfat, adrenal, liver and pancreas. Within the tissues, the radioactivitywas mainly distributed in the TG and/or phospholipid (PL) fractions.¹⁴C-EPA-E was hydrolyzed rapidly in the small intestine homogenates,lymph, plasma and liver homogenates of the rat. Orally administeredEPA-E was primarily incorporated in adrenal gland lipids as cholesterolesters of the fatty acids eicosapentaenoic acid (EPA), docosapentaenoicacid (DPA) and docosahexaenoic acid (DHA).

The uptake of ¹⁴C-EPA-E-derived radioactivity in rats described above iscompatible with the absorption process of essential fatty acids inanimals and humans described below. In addition, esterases are alsodistributed in most of the organs in humans). Therefore, the absorptionand distribution profiles after oral dosing of EPA-E should bequalitatively similar in humans and dogs as observed in rats.

The digestion and absorption of essential fatty acids, mainly in theform of TG, are known to involve several processes.

-   -   The fatty acids are rapidly hydrolyzed from the TG to FFA by        lipase in the intestine.    -   The FFA are taken up by the enterocytes where they are        re-esterified into TG and enter the blood circulation, mainly        through the lymphatic route as chylomicrons.    -   In the tissues, the TG of the chylomicrons is hydrolyzed again        by lipoprotein lipase to FFA and taken up by the tissues.

Consequently, when EPA-E is administered orally to humans, it wouldappear that it is well absorbed, even though the unchanged ethyl esterform is not detected, and the free form (EPA) is only detected at a verylow level, in the blood. Due to this absorption mechanism of essentialfatty acids, the administered EPA-E exists as a blood-constituent fattyacid in the total lipids. In fact, according to the approval packagedocument of Lovaza®, the free form of EPA is undetectable in thecirculation (<1 μM) following an oral dose of 4 g of Omacor® (a mixtureof the ethyl esters of EPA and DHA) Following oral administration ofEPA-E, EPA, DPA and DHA were isolated and identified as metabolites intissues and plasma. EPA, DPA and DHA were incorporated into TG and PL.Radioactivity was primarily excreted in expired air (44%) after singleoral administration at 30 mg/kg EPA-E with minimal excretion in bile andurine (˜3%) and approximately 20% excreted in feces. Thus, therespiratory route (as ¹⁴CO2) was considered to be the major eliminationpathway of ¹⁴C-EPA-E. Excretion of radioactivity in dogs after singleoral administration at 30 mg/kg was low with 1.0% excreted in the urineand 19.2% recovered in the feces after 1 week.

Renal excretion was the elimination route for several minor highly polarmetabolites (<0.4%), but EPA-E, EPA, DPA or DHA were not detected in theurine. In feces, EPA-E and EPA, evidently derived from the unabsorbeddrug, were detected; however, no DPA or DHA was detected in the feces.

In metabolism studies conducted in vitro, when ¹⁴C-EPA-CoA was incubatedwith the rat liver mitochondrial fraction, 17.1% of the radioactivityadded to the incubation mixture was detected as ¹⁴CO2 and the formationof carbon chain-shortened products was observed. Incubation of¹⁴CEPA-CoA with a rat peroxisome fraction also resulted in the formationof carbon chain-shortened products. These results show that EPA, afterbeing taken up by the rat tissues, is finally almost entirely oxidizedto CO2 by mitochondrial and peroxisomal (3-oxidation.

After oral administration of ¹⁴C-EPA-E to the rat, EPA, DPA and DHA weredetected as metabolites in the total lipid fraction of the plasma andtissues (liver, fat, heart and brain). However, no unchanged EPA-E wasfound in the plasma or any of the tissues. In the total lipid fractionof the liver, the radioactivity originated mainly from EPA, DPA and DHA.Thus, it is considered that EPA, DPA and DHA are the predominantmetabolites of ¹⁴C-EPA-E in tissues, as constituent fatty acids of thetotal lipid fraction. When ¹⁴C-EPA-K was incubated with the microsomalfraction, formation of DPA and DHA was detected. These results show thatEPA taken up by the rat tissues is elongated to DPA and DHA in themicrosomes.

Plasma protein binding in rats and dogs was >86% and >96%, respectively.Previous studies have shown that EPA is unlikely to inhibit CYP450 atfree concentrations observed in humans.

Example 2 Clinical Trial Data Study Design

This example provides the protocol used for an ongoing phase II clinicaltrial, double blind, placebo-controlled study to investigate the safety,efficacy, and pharmacokinetic profile of two doses of EPA-E in subjectswith NASH. Up to 70 subjects were enrolled into each treatment arm, fora total of 210 subjects to be enrolled. Block randomization using aninteractive voice response system (IVRS) was used to assign patients ina 1:1:1 ratio to two active doses and placebo. Patients were stratifiedat randomization by presence or absence of diabetes. Patients withdiabetes comprised no more than 25% of the total number of patientsenrolled. Subjects were treated with 600 mg EPA-E, 900 mg EPA-E orplacebo three times a day for one year.

-   -   Study arm 1: 600 mg EPA-E (3 capsules), TID    -   Study arm 2: 900 mg EPA-E (3 capsules), TID    -   Study arm 3: placebo (3 capsules), TID

Subjects were required to have a liver biopsy with proven NASH in the 6month period prior to screening. The Pharmacokinetic profile for EPA-Ewas evaluated in a subgroup of subjects in specified sites. Subjectswere approached prior to providing informed consent to determine if theywill participate in the PK group subset. Approximately 36 subjectsparticipated in the PK subset evaluation (12 from each treatment arm, toinclude 6 males and 6 females). A study schematic is provided in FIG. 6.Overall study duration plan was 2 years.

The Investigational Drugs

In order to support the trial, EPA-E capsules and matching placebocapsules were prepared. EPA-E capsule is an oval soft gelatin capsulecontaining 300 mg of EPA-E as an active ingredient. Placebo capsule isan oval soft gelatin capsule containing olive oil as an inactiveingredient. These capsules are unidentifiable as EPA-E capsules orplacebo capsules.

Subjects were administered orally 3 capsules 3 times daily, immediatelyafter meals.

Eligibility Criteria

Subjects with a histological diagnosis of NASH are eligible.

Inclusion Criteria

Inclusion criteria were designed to ensure that subjects with biopsyproven NASH are included, and to avoid situations where potential harmmay occur to subjects in conjunction with participation in the study.

Subjects were potentially included into the study if they met all thefollowing criteria:

1. Diagnosis of definite NASH by the central reading pathologists:

-   -   Liver biopsy slides will be submitted for evaluation by the        central pathologists according to one of the following criteria:    -   a. Previous liver biopsies must have been obtained within 6        months prior to informed consent and should be judged by the        local pathologist as showing NAS >4 with a minimum score of 1        each for steatosis and lobular inflammation plus EITHER        ballooning OR at least la sinusoidal fibrosis AND a finding of        possible or definite steatohepatitis    -   b. For liver biopsies performed after informed consent is        obtained, all slides will be submitted for reading by the        central pathologists in conformity with the independent        pathology review charter (IPRC)

2. Patients of either gender greater than 18 years of age

3. Patients with diabetes that have been on stable doses ofanti-diabetic agents since at least 6 months prior to liver biopsy maybe enrolled

4. Females must be of non-child bearing potential (surgically sterilizedor at least two years post-menopausal) or if of child-bearing potential,must have a negative pregnancy test at screening and agree to use aneffective form of contraception during the study and for at least 30days following the last dose of study medication

5. Normal ECG or clinically non-significant findings at the Screeningand Baseline visits

6. No significant concomitant medical illness, without any clinicallysignificant physical exam findings and without any clinicallysignificant laboratory findings, as determined by the principalinvestigator

7. Signed an informed consent form indicating that they understand thepurpose of and procedures required for the study and are willing toparticipate in the study and comply with the study procedures andrestrictions

Exclusion Criteria

Exclusion criteria have been designed to exclude subjects from the studyif they will not be evaluable for the primary endpoint, if they havedisease states that would interfere with analysis of study endpoints, orwould put subjects at risk of serious adverse events associated withtheir participation.

Potential subjects will be excluded from participating in the study ifthey meet any of the following exclusion criteria:

1. Inability or unwillingness to have a liver biopsy

2. Diagnosis of cirrhosis by central pathology reviewers

3. Previous bariatric surgery or biliary diversion (i.e. gastricbypass), esophageal banding and gastric banding

4. Serum ALT >300 U/L

5. Subject has used drugs associated with steatohepatitis within 6months prior to screening (corticosteroids, high dose estrogens,methotrexate, amiodarone, anti-HIV drugs, tamoxifen, diltiazem)

6. Use of the following anti-NASH agents for more than a 2 week periodin the 3 months prior to liver biopsy or the 3 months prior toscreening:

-   -   a. Vitamin E >60 IU per day    -   b. Omega-3-acid ethyl esters or omega-3-PUFA-containing        supplements >200 mg per day    -   c. Thiazoledinediones (e.g. pioglitazone)

7. Patients on a non-stable dose of the following anti-NASH agentswithin 6 months of the liver biopsy or within 6 months of the screeningvisit: HMG-CoA reductase inhibitors (statins), fibrates, probucol,ezetimibe, ursodiol (UDCA), taurine, betaine, Nacetylcysteine,s-adenosylmethionine (SAM-e), milk thistle, anti-TNF therapies, orprobiotics

8. Greater than a 10% decrease in weight within 8 weeks of baselinevisit

9. Alcohol consumption >30 g/day, currently or for more than 3consecutive months within 5 years of screening

10. Blood alcohol level greater than 0.02% at screening and/or baseline

11. Evidence of active substance abuse, including prescription andrecreational drugs

12. Other liver disease (hepatitis C, hepatitis B, Wilson's, autoimmune,α-1-antitrypsin and hemochromatosis) or known HIV infection

13. Pregnant or lactating at the screening visit

14. Renal insufficiency (creatinine >2 mg/dL), symptomatic coronary,peripheral or neurovascular disease, symptomatic heart failure (NYHA2-4) or advanced respiratory disease requiring oxygen therapy

15. History of cerebral or retinal hemorrhage or other bleedingdiathesis

16. QTc >450 msec for males and >470 for females as corrected by theFridericia formula

17. Inability to provide written informed consent

18. Received any investigational agent or participation in any clinicalstudy of an investigational agent or investigational therapy within 3months prior to the screening visit.

19. Any condition in the opinion of the Principal Investigator thatwould contraindicate the patient's participation

Prohibited and Concomitant Medications

All prescription and over-the-counter medications taken by subjectsduring the 30 days before screening up to the start of treatment wererecorded. The following medications were prohibited during participationin the study:

-   -   Omega-3-acid ethyl esters and omega-3-PUFA containing        supplements >200 mg per day    -   Vitamin E >60 IU per day    -   Thiazolidinediones (e.g. pioglitazone)

The following medications were allowed during the study according to thespecified restrictions:

-   -   Subjects could continue prescription or over-the-counter        medications or herbal remedies (HMG-CoA reductase inhibitors        [statins], fibrates, probucol, ezetimibe, ursodiol [UDCA],        taurine, betaine, N-acetylcysteine, s-adenosylmethionine        [SAM-e], milk thistle, anti-TNF therapies, or probiotics) ONLY        if they have been on a stable dose for at least 6 months prior        to screening    -   Subjects could continue the following anti-diabetic medications        if they have been taking stable doses since at least 6 months        prior to liver biopsy: biguanides (metformin), insulin,        sulfonylureas, alpha-glucosidase inhibitors (acarbose), and        phenylalanine derivatives (nateglinide)    -   Any subjects receiving anti-platelet therapy or anti-thrombotic        agents (e.g. warfarin, ASA, and clopidogrel) after study        commencement should be monitored closely during the study

Preliminary Results

A) A sub-population of NASH patients were counted and classified asresponders or non responders as shown in FIG. 1. Preliminary dataindicated higher efficacy of the pharmaceutical composition in patientswho were determined not to have diabetes or showed mild diabetes ascompared to diabetic patients in Study arm 1 (600mg EPA-E, TID). Theefficacy of Study arm 2 (900mg EPA-E, TID) was also equivalent to thatof Study arm 1.

The same sub-population of patients was further characterized. HbA1clevels were measured for patients, whose HbA1c=<6.4, as shown in FIG. 2.Preliminary data indicates higher efficacy of the pharmaceuticalcomposition in patients with HbA1c levels =<6.4 in all cases, andespecially who would be classified as non-diabetic(includingpre-diabetic) based on guidelines as set by the ADA and as describedherein in Study arm 1. The efficacy of Study arm 2 was also equivalentto that of Study arm 1.

Diabetes in the same sub population of patients was alternativelymeasured using a test for fasting glucose. Glucose levels were measuredfor patients, whose glucose levels were =<125 mg/dL and, as shown inFIG. 3. Preliminary data indicates higher efficacy of the pharmaceuticalcomposition in patients with =<125 mg/dL and who would be classified asnon-diabetic(including pre-diabetic) or diabetic based on guidelines asset by the ADA and as described herein in Study arm 1. The efficacy ofStudy arm 2 was also equivalent to that of Study arm 1.

The chart in FIG. 4 indicates that taken together,non-diabetic(including pre-diabetic) or diabetic, as measured by bothfasting glucose=<125 mg/dL and HbA1c=<6.4, indicated a higher responseto rate to the EPA-E in Study arm 1. The efficacy of Study arm 2 wasalso equivalent to that of Study arm 1.

B) A second sub-population of NASH patients were counted and classifiedas responders or non responders as shown in FIG. 7. Preliminary dataindicates higher efficacy of the pharmaceutical composition in patientswho were determined not to have biliary tract disease markers associatedwith the disease in both Study arm 1 (600mg EPA-E, TID) and Study arm 2(900mg EPA-E, TID).

γ-Glutamyl Transferase (GGT) v. Preliminary Endpoint achievement rate isreflected in FIG. 8. Preliminary data indicates higher efficacy of thepharmaceutical composition in patients with GGT levels=<60 IU/L in bothStudy arm 1 and 2.

The same sub-population of patients was further characterized. NASHresponse (change of NAS score) was measured for patients, whose GGTlevels=<33 IU/L, as shown in the table of FIG. 9. Preliminary dataindicates higher efficacy of the pharmaceutical composition in patientswith GGT levels=<33 IU/L, and who would be classified as not havingbiliary duct disease in both Study arm 1 and 2.

Alternatively, improvement of serum EPA/AA ratio on Day 365 of patientswith γ-Glutamyl Transferase (GGT) levels≤33 IU/L was also measured, asshown in the table of FIG. 10. Preliminary data indicates higherefficacy of the pharmaceutical composition in patients with ≤33 IU/L andwho would be classified as not biliary duct disease in Study arm 2.

The table in FIG. 11 provides corresponding reference values forparameters of liver function in patients with γ-Glutamyl Transferase(GGT) levels ≤33 IU/L and patients with γ-Glutamyl Transferase (GGT)levels ≥33 IU/L. Serum direct bilirubin levels were also assays in thesub group of patients with γ-Glutamyl Transferase (GGT) levels ≥33IU/L/L. All patients were observed within reference (normal) value fordirect bilirubin levels. The range of serum direct bilirubin of patientswith serum GGT levels ≤33 IU/L was between 0.03 and 0.17.

FIG. 12 is a chart representing serum EPA/AA ratios for variousadministered dosages of EPA-E over time. The chart indicates asignificant increase in EPA/AA ratio in patients with γ-GlutamylTransferase (GGT) levels ≤33 IU/L in Study arm 2. The range of theimprovement in this ratio on Day 365 of patients with serum GGT levels≤33 IU/L was observed between 0.0406 and 1.637 for administered 2700mg/day dose of EPA-E.

ALP levels were also assays in the sub group of patients with γ-GlutamylTransferase (GGT) levels ≤33 IU/Land patients with γ-GlutamylTransferase (GGT) levels ≥33 IU/L. Some patients in GGT>=33 IU/L andplacebo group showed ALP value above reference (normal) value as shownin FIG.

13.

Example 3 Self-Emulsifying Formulation

0.5 g of soybean lecithin, 1.0 g of polyoxyethylene (60) hydrogenatedcastor oil, 0.4 g of propylene glycol, and 3.1 g of EPA₌E were weighed,and mixed while heating to a temperature of about 70° C. to prepare aself-emulsifying composition. After substituting with nitrogen, theself-emulsifying composition was hermetically sealed and stored at roomtemperature until the evaluation. Formulation of the self-emulsifyingcomposition is shown below:

Ingredients Formulation (% by weight) EPA-E 62.0 Soybean lecithin 10.0Polyoxyethylene (60) hydrogenated castor oil 20.0 Propylene glycol 8.0Total 100.0

Example 4 Self-Emulsifying Formulation

0.5 g of soybean lecithin, 1.0 g of polyoxyethylene (50) hydrogenatedcastor oil, 0.4 g of propylene glycol, and 3.1 g of EPA-E were weighed,and a self-emulsifying composition was prepared and stored by repeatingthe procedure of Example 3. Formulation of the self-emulsifyingcomposition is shown below:

Ingredients Formulation (% by weight) EPA-E 62.0 Soybean lecithin 10.0Polyoxyethylene (60) hydrogenated castor oil 20.0 Propylene glycol 8.0Total 100.0

Example 5 Self-Emulsifying Formulation

0.5 g of soybean lecithin. 0.9 g of polyoxyethylene castor oil, 0.6 g ofpropylene glycol, and 3.0 g of EPA-E were weighed, and aself-emulsifying composition was prepared and stored by repeating theprocedure of Example 1. Formulation of the self-emulsifying compositionis shown below:

Ingredients Formulation (% by weight) EPA-E 60.0 Soybean lecithin 10.0Polyoxyethylene castor oil 18.0 Propylene glycol 12.0 Total 100.0

Example 6 Self-Emulsifying Formulation

0.6 g of soybean lecithin, 0.6 g of polyoxyethylene (60) hydrogenatedcastor oil, 0.5 g of propylene glycol, and 3.3 g of EPA-E were weighed,and a self-emulsifying composition was prepared and stored by repeatingthe procedure of Example 1. Formulation of the self-emulsifyingcomposition is shown below:

Ingredients Formulation (% by weight) EPA-E 66.0 Soybean lecithin 12.0Polyoxyethylene (60) hydrogenated castor oil 12.0 Propylene glycol 10.0Total 100.0

Example 7 Self-Emulsifying Formulation

0.5 g of soybean lecithin, 0.5 g of polyoxyethylene (50) hydrogenatedcastor oil, 0.5 g of propylene glycol, and 3.5 g of EPA-E were weighed,and a self-emulsifying composition was prepared and stored by repeatingthe procedure of Example 3. Formulation of the self-emulsifyingcomposition is shown below:

Ingredients Formulation (% by weight) EPA-E 70.0 Soybean lecithin 10.0Polyoxyethylene (50) hydrogenated castor oil 10.0 Propylene glycol 10.0Total 100.0

Example 8 Self-Emulsifying Formulation

0.3 g of soybean lecithin, 0.3 g of polyoxyethylene (20) sorbitanmonooleate, 0.9 g of polyoxyethylene (60) hydrogenated castor oil, 0.4 gof propylene glycol, and 3.1 g of EPA-E were weighed, and aself-emulsifying composition was prepared and stored by repeating theprocedure of Example 3. Formulation of the self-emulsifying compositionis shown below:

Ingredients Formulation (% by weight) EPA-E 62.0 Soybean lecithin 6.0Polyoxyethylene (20) sorbitan monooleate 6.0 Polyoxyethylene (60)hydrogenated castor oil 18.0 Propylene glycol 8.0 Total 100.0

What is claimed is:
 1. A method for treating a fatty liver disorder in ahuman subject in need thereof, comprising administering atherapeutically effective amount of a pharmaceutical composition thatcomprises eicosapentaenoic acid (EPA), ethyl eicosapentanoate (EPA-E),or a pharmaceutically acceptable salt thereof to the subject, whereinthe subject has a serum gamma glutamyl transferase (GGT) activity levelhigher than 33 IU/L before the administering, and wherein theadministering results in a reduction of 1% to about 90% of alkalinephosphatase (ALP) concentration in the subject.
 2. The method of claim1, wherein the subject's serum HbA1c level is equal to or less than 6.4%or the subject's fasting serum glucose level is equal to or less than125 mg/dl, before the administering.
 3. The method of claim 1, whereinthe administering further results in an improvement of serumeicosapentaenoic acid/arachidonic acid (EPA/AA) ratio, as compared to abaseline EPA/AA ratio, of equal to or greater than 0.4 in the subject.4. The method of claim 1, wherein the subject is not treated with ananti-diabetic agent.
 5. The method of claim 1, wherein the administeringis in combination with administering one or more anti-diabetic agents.6. The method of claim 1, wherein the subject has a: i) non-alcoholicfatty liver disease activity score (NAS) greater than or equal to 3; orii) steatosis score equal to or greater than 1; or iii) lobularinflammation score equal to or greater than 1; or iv) ballooning scoreequal to or greater than 1; or v) fibrosis score equal to or greaterthan
 1. 7. The method of claim 6, further comprising improving asteatosis and lobular inflammation condition of the subject; wherein thesubject exhibits, as compared to a baseline pretreatment level, at least1% reduction for: alanine aminotransferase (ALT), aspartateaminotransferase (AST), triglyceride (TG), TG/High-density lipoprotein(HDL) ratio, Free fatty acid, arachidonic acid (AA), monounsaturatedfatty acid (MUFA), Palmitoleic acid, Oleic acid, Oleic acid/Stearic acidratio, Palmitoleic acid/Palmitic acid ratio, Stearic acid/Palmitic acidratio, γ-linoleic acid/Linoleic acid ratio, Adrenic acid/AA ratio,Ferritin, Thioredoxin, Tumor necrosis factor (TNF a), Soluble TumorNecrosis Factor Receptor 1 (sTNF-R1), Soluble Tumor Necrosis FactorReceptor 2 (sTNF-R2), High Sensitivity C-reactive protein (Hs-CRP),Connective Tissue Growth Factor (CTGF), Soluble Cluster ofDifferentiation 40 (sCD40), Leptin, complement factor D, cytokeratin 18(CK18) fragment, serum High Mobility Group Box 1 (HMGB1), Fas,procollagen III peptide or PAI-1; at least 5% increase for EPA or EPA/AAratio; at least 1% increase for Docosapentaenoic acid (DPA), orAA/Homo-γ-linoleic acid ratio; or no worsening of ALP, bilirubin, HighDensity Lipoprotein Cholesterol (HDL-C), Low Density LipoproteinCholesterol (LDL-C), Total Cholesterol (TC), non-HDL-C, HomeostaticModel Assessment Insulin Resistance (HOMA-IR), Glucose, Fasting plasmaglucose, postprandial plasma glucose, Oral Glucose Tolerance Test(OGTT), platelet count or Body Mass Index (BMI).
 8. The method of claim5, wherein the anti-diabetic agent is selected from the following group:peroxisome proliferator-activated receptor gamma (PPARγ) agonists,biguanides, protein tyrosine phosphatase-1B (PTP-1B) inhibitors,meglitinides, a glucoside hydrolase inhibitors, insulin secreatagogues,A2 antagonists, insulin and related compounds, non-thiazolidinediones,glycogen synthase kinase 3β (GSK 3β)/glycogen synthase kinase 3 (GSK 3)inhibitors, dipeptidyl peptidase IV (DPP-IV) inhibitors, peptides,sulfonylureas, and nonsulfonylurea secretagogues.
 9. The method of claim1, wherein the fatty liver disorder is selected from the groupconsisting of Non-Alcoholic Fatty Liver Disease (NAFLD), andNon-Alcoholic Steatohepatitis (NASH).
 10. The method of claim 1,comprising administering the EPA-E to the subject in an amount betweenabout 1800 and about 2700 mg per day.
 11. The method of claim 1,comprising administering to the subject at least 1800 mg of the EPA-Eper day.
 12. The method of claim 1, comprising administering to thesubject at least 2700 mg of the EPA-E per day.
 13. The method of claim1, wherein the subject is further characterized by having at least onecondition selected from the group consisting of high triglycerides, lowHDL-C, impaired glucose tolerance, and impaired metabolic syndrome. 14.The method of claim 7, wherein the subject exhibits, as compared to abaseline pretreatment level, at least 5% reduction in ALT and/or AST.15. The method of claim 1, further comprising determining in the subjectprior to treatment a baseline level in serum of at least one memberselected from the group consisting of ALT in a range of 10 to 300 IU/L,AST in a range of 10 to 250 IU/L, HDL-C in a range of 25 to 55 mg/dl,LDL-C in a range of 100 to 200 mg/dl, triglycerides in a range of 100 to1000 mg/dl, TC in a range of 170 to 300 mg/dl, High TG, low HDL-C,TG/HDL-C ratio in a range of 3.75 to 10, non-HDL-C in a range of 100 to250 mg/dl, Free fatty acid in a range of 400 to 1000 μEq/L, HOMA-IR in arange of 1.5 to 5, HbA1c in a range of 5.7 to 10%, and Fasting plasmaglucose in a range of 100 to 200 mg/dl.
 16. The method of claim 1,wherein the pharmaceutical composition is administered to the subject 1to 4 times per day.
 17. The method of claim 1, wherein thepharmaceutical composition is a self-emulsifying composition thatcomprises 50 to 95% by weight of the EPA-E.
 18. The method of claim 1,wherein the pharmaceutical composition comprises 5 to 50% by weight ofan emulsifier having a hydrophilic lipophilic balance of at least 10.19. The method of claim 1, wherein the pharmaceutical compositioncomprises ethanol, the content of which is up to 4% by weight inrelation to the total content of the self-emulsifying composition. 20.The method of claim 1, wherein the pharmaceutical composition comprisesan emulsifier that is at least one member selected from the groupconsisting of polyoxyethylene hydrogenated castor oil, polyoxyethylenesorbitan fatty acid ester, polyoxyethylene castor oil, polyethyleneglycol fatty acid ester, polyoxyethylene polyoxypropylene glycol,sucrose fatty acid ester, and lecithin.
 21. The method of claim 1,wherein the pharmaceutical composition comprises a lecithin.
 22. Themethod of claim 1, wherein the pharmaceutical composition comprises apolyhydric alcohol.
 23. The method of claim 1, wherein thepharmaceutical composition further contains at least one member selectedfrom the group consisting of docosahexaenoic acid, a pharmaceuticallyacceptable salt, and a pharmaceutically acceptable ester thereof. 24.The method of claim 1, wherein the pharmaceutical composition comprisesthe EPA.
 25. The method of claim 1, wherein the pharmaceuticalcomposition further comprises ethyl docosahexaenoate.
 26. The method ofclaim 1, wherein the pharmaceutical composition comprises an emulsifier,and wherein the total content of the emulsifier is 10 to 100 parts byweight in relation to 100 parts by weight of at least one compoundselected from the group consisting of ω3 polyunsaturated fatty acids andtheir pharmaceutically acceptable salts and esters in the pharmaceuticalcomposition.
 27. The method of claim 1, wherein EPA-E present in thepharmaceutical composition is at least 40% by weight in total of fattyacids and derivatives thereof.
 28. The method of claim 1, wherein thepharmaceutical composition-comprises the EPA-E.
 29. The method of claim1, wherein the subject is non diabetic, pre-diabetic, or mildlydiabetic.